Annals of Clinical & Laboratory Science, vol. 31, no. 1, 2001 25

Review: Mitochondrial Medicine – Molecular of Defective Oxidative Phosphorylation

Egil Fosslien Department of Pathology, College of Medicine, University of Illinois at Chicago, Chicago, Illinois

Abstract. Different tissues display distinct sensitivities to defective mitochondrial oxidative phosphorylation (OXPHOS). Tissues highly dependent on oxygen such as the cardiac muscle, skeletal and smooth muscle, the central and peripheral nervous system, the kidney, and the insulin-producing pancreatic β-cell are especially susceptible to defective OXPHOS. There is evidence that defective OXPHOS plays an important role in atherogenesis, in the of Alzheimer’s , Parkinson’s disease, diabetes, and aging. Defective OXPHOS may be caused by abnormal mitochondrial biosynthesis due to inherited or acquired mutations in the nuclear (n) or mitochondrial (mt) deoxyribonucleic acid (DNA). For instance, the presence of a mutation of the mtDNA in the pancreatic β-cell impairs adenosine triphosphate (ATP) generation and insulin synthesis. The nuclear genome controls mitochondrial biosynthesis, but mtDNA has a much higher mutation rate than nDNA because it lacks histones and is exposed to the radical oxygen species (ROS) generated by the electron transport chain, and the mtDNA repair system is limited. Defective OXPHOS may be caused by insufficient fuel supply, by defective electron transport chain enzymes (Complexes I - IV), lack of the electron carrier coenzyme Q10, lack of oxygen due to or anemia, or excessive membrane leakage, resulting in insufficient mitochondrial inner membrane potential for ATP synthesis by the F0F1-ATPase. Human tissues can counteract OXPHOS defects by stimulating mitochondrial biosynthesis; however, above a certain threshold the lack of ATP causes . Many agents affect OXPHOS. Several nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit or uncouple OXPHOS and induce the ‘topical’ phase of gastrointestinal ulcer formation. Uncoupled mitochondria reduce cell viability. The Helicobacter pylori induces uncoupling. The uncoupling that opens the membrane pores can activate . Cholic acid in experimental atherogenic diets inhibits Complex IV, cocaine inhibits Complex I, the poliovirus inhibits Complex II, ceramide inhibits Complex III, azide, cyanide, chloroform, and methamphetamine inhibit Complex IV. Ethanol abuse and antiviral nucleoside analogue therapy inhibit mtDNA replication. By contrast, melatonin stimulates Complexes I and IV and Gingko biloba stimulates Complexes I and III. Oral Q10 supplementation is effective in treating cardiomyopathies and in restoring plasma levels reduced by the statin type of cholesterol-lowering drugs. (received 15 September 2000; accepted 30 October 2000)

Keywords: mitochondria, oxidative phosphorylation, apoptosis, atherosclerosis, diabetes mellitus, gastrointestinal disease, Alzheimer’s disease, Parkinson’s disease, Helicobacter pylori, ethanol, statin, melatonin, .

Introduction (DNA, lipids, proteins) [1]. Mitochondrial medicine was initiated six years later with the report on Luft’s Historical perspective, In 1956 Harman introduced syndrome, a mitochondrial functional disorder the free radical theory of aging suggesting that over involving loose coupling and a greatly elevated basic time, free radicals cause damage to macromolecules metabolic rate despite normal thyroid [2]. In 1963, threadlike structures that could be digested Address correspondence to Egil Fosslien, M.D., Department of by DNAase were discovered in mitochondria of chick Pathology (M/C 847), College of Medicine, University of Illinois at Chicago, 1819 West Polk Street, Chicago IL 60612; tel 312 embryos [3,4]. The following year, mtDNA was 996 7323; fax 312 996 7586, e-mail [email protected] isolated from purified yeast mitochondria. Phosphory-

0091-7370/01/0000-0025 $10.75; © 2001 by the Association of Clinical Scientists, Inc. 26 Annals of Clinical & Laboratory Science lating submitochondrial particles were first isolated triphosphate (ATP), the chemical energy required for from yeast in 1966 [5]. a cell’s metabolism [18]. Therefore, each step of the In 1970, the first respiratory chain deficiency mitochondrial energy conversion system must function without elevated basal oxygen consumption that was efficiently for cell survival. Auspiciously, it is a due to a reduced activity of a specific enzyme, redundant system, since each cell carries up to a few cytochrome b, was identified. The symptoms included thousand mitochondria. Furthermore, when a cell with dementia, cerebellar ataxia, and proximal muscle a few defective mitochondria divides, one daughter cell weakness [6]. Next came reports on a defect of pyruvate might receive the defective mitochondria and perish, dehydrogenase [7] and two years later the identification while the other daughter cell is restored to normal. of reduced cytochrome oxidase c in Menkes’ syndrome However, in post-mitotic cells this option is not [8]. Another year later, myopathies due to a defect in available. It has been estimated that over 90% of mito- muscle carnitine [9] and defects in carnitine palmitoyl chondria are contained in postmitotic cells [19]. transferase were reported [10]. Because of differences in the threshold values of In 1981, the first complete (Cambridge) sequence different tissues, only some tissues may be affected and of mitochondrial DNA (mtDNA) was published [11]. exhibit pathology as a result of an inherited mitochon- In 1984, Miquel and Fleming emphasized that the drial DNA mutation, even if that mutation is present mitochondrion is the major site of oxidative damage in all tissues [20]. Similarly, exogenous substances that and that oxidative damage to mitochondria plays an affect OXPHOS may affect different tissues differently. important role in aging [12]. The first neurodegen- erative disease associated with a mutation of mtDNA Classification of Defects was described in 1988 [13]. The following year Linnane et al expanded on the Structural defects. 1. Inherited. Primary mitochon- mitochondrial theory of aging: somatic accumulation drial are caused by inherited defects in the of mitochondrial genome mutations during life is a mitochondrial structure, which lead to defective major cause of human aging and degenerative disease function of the mitochondria. They involve oxidative [14]. Their theory was based upon the following phosphorylation or other mitochondrial pathways like observations: (a) high frequency of gene mutation in the urea cycle and fatty acid oxidation [21]. They cause mitochondrial DNA, (b) the small size of the a wide range of diverse clinical manifestations; such as mitochondrial genome, (c) lack of effective repair ataxia, cardiomyopathy, dementia, epilepsy, myopathy, mechanism for mtDNA, and (d) somatic segregation polyneuropathy, and retinal pigment anomaly [22,23]. of individual mtDNA during eukaryotic cell division. 2. Acquired. (a) Ischemia and deletion. In ischemic Mitochondrial DNA damage may be a biomarker heart disease, a 40-45-fold increase in the common of oxygen damage over time. Mutations in mtDNA, (4,799 bp) mtDNA deletion in the myocardium has whether inborn or acquired, accelerate mitochondrial been reported. Transcripts of mtDNA-coded oxidative dysfunction that induces further damage, and phosphorylation enzyme subunits were increased, additional mutations and deletions [15]. pointing to an association between ischemia and Presently, it is realized that a gradual decline in ATP OXPHOS deficiency, enhanced ROS generation and generation with age induces loss of stamina, memory, mtDNA deletions [24]. Furthermore, animal models vision, and hearing and contributes to aging-related point to ischemia as the primary event. For instance, diseases such as cardiovascular disease and diabetes [16]. myocardial ischemia produced by ameroid constriction In addition, many exogenous agents, even therapeutic of canine coronary arteries resulted in the development ones such as acidic nonsteroidal anti-inflammatory of myocardial mtDNA deletions similar to deletions drugs (NSAIDs), can inhibit the mitochondrial energy observed in human ischemic hearts [25]. system [17] and cause disease at any age. (b) Organ preservation. Prevention of serious impairment of oxidative phosphorylation from Oxidative phosphorylation. An efficient mitochondrial ischemia reperfusion injury is critical to transplantation oxidative phosphorylation (OXPHOS) system is and open heart surgery. It has been suggested that essential since it provides most of the adenosine lipid peroxidation plays a role in the deterioration in of the mitochondrion 27 mitochondrial oxidative phosphorylation observed in carried via coenzyme-Q10 (Q10) to Complex III, and the isolated rat heart after 8-12 hr of hypothermic then via cytochrome c and Complex IV to oxygen. ischemia [26]. An ample supply of oxygen as electron acceptor must be provided. Uninhibited, effective proton pumping Functional defects. This intricate energy conversion by Complexes I, III, and IV must deposit the released system is susceptible to malfunction by many causes energy as an electrical charge across the mitochondrial occurring either alone or in various combinations. A inner membrane. Salicylate and indomethacin can few causes are illustrated in Figure 1. Many NSAIDs inhibit the electron transport chain. The electron that inhibit oxidative phosphorylation inhibit the transport may be reduced or blocked by an inadequate enzyme activities rather than their synthesis. supply of oxygen. Blood flow reduction due to 1. Inadequate fuel supply. The generation of the atherosclerosis, reduction in oxygen carrying capacity mitochondrial membrane potential requires an due to anemia or smoking, or a reduction in the oxygen adequate supply of metabolites to Complexes I and II partial pressure at high altitude or reduced airplane of the electron transport chain. For instance, aspirin cabin pressure can all impair the function of the inhibits β-oxidation and the delivery of metabolites to electron transport chain. Furthermore, defective ATP the electron transport chain [27] and reduces the generation may be due to lack of substrates, essential mitochondrial fuel supply and energy flux. It has been cofactors, or may be caused by inefficient use of oxygen implied that chronic inhibition of β-oxidation causes [29]. A virus [30,31] may induce OXPHOS hepatic by choking the respiratory chain and dysfunction. Conditions of oxidative stress, chronic thus increasing the production of reactive oxygen alcohol abuse, a large number of drugs, or toxin can species. These effects might lead to lipid peroxidation also lead to a decline of mitochondrial function [21]. that causes steatohepatitis and the formation of Mallory bodies, , and fibrosis [28]. 3. Abnormal coupling and uncoupling. The inner 2. Dysfunction of the electron transport chain. mitochondrial membrane potential serves as a Electrons from the metabolites must be efficiently convertible energy currency of the cell [32] (Figure 1).

Fig. 1. Illustration shows defective mitochondrial oxidative phosphorylation (OXPHOS). Numbers in brackets refer to numbers in square boxes. Defective OXPHOS impairs the electron flow from metabolites (fuel) [1] through the electron transport chain [2] (enzyme Complexes I - IV) to oxygen. The generation of the inner mitochondrial membrane potential ∆Ψ ( m) [3] is lowered and phosphorylation [4] reduced, leading to insufficient generation of adenosine triphosphate (ATP) by F0F1-ATPase (OXPHOS complex V) for proper cellular function. Inhibition of any of the enzyme complexes of oxidative ∆Ψ phosphorylation (for example by an acid NSAID) lowers m generation. Ischemia caused by atherosclerosis or anemia reduces respiration. Inhibition of phosphorylation or loss of potential without ATP generation by passive or induced membrane proton leaks causes uncoupling of oxidative phosphorylation. During the mitochondrial permeability transition (MPT) mitochondrial pores open, causing membrane depolarization, loss of membrane potential, cessation of oxidative phosphorylation, and initiation of apoptosis. The text in italics indicates exogenous sources of mitochondrial dysfunction. 28 Annals of Clinical & Laboratory Science

Therefore, the majority of the stored energy must be oxygen species increases and α-tocopherol is depleted efficiently coupled to Complex V to phosphorylate probably because of consumption due to lipid adenosine diphosphate (ADP) to adenosine triphos- peroxidation [40]. phate (ATP). Uncoupling in the form of inhibition of 2. Antioxidants. Experimentally, the oxidative phosphorylation must be limited. Uncoupling in the stress can be enhanced by gene inactivation of the form of losses due to passive membrane permeability, antioxidant enzyme glutathione peroxidase-1 (Gpx1) altered membrane lipids, or uncoupling proteins must in the mutant mouse. It is normally highly expressed be controlled. The amount of heat generated when in the mouse liver. The presence of the mutated Gpx1- some hydrogen ions passively leak back across the gene causes a significant increase in hydrogen peroxide membrane must be restricted, and the heat released by release by the liver mitochondria. Moreover, it active channeling of protons back through the markedly degrades the efficiency of mitochondrial membrane by uncoupling proteins (UCP) must be oxidative phosphorylation as evidenced by a significant efficiently regulated. Induction of the membrane reduction in the respiratory control ratio [41]. permeability transition (MPT) by NSAIDs (eg, diclo- fenac sodium,mefenamic acid [33]), or by toxic Mitochondrial Dysfunction in Major Diseases endogenous or exogenous agents, must be limited. As an example of membrane leakage, in Luft’s Cardiovascular diseases. 1. Early suggestions of disease the loose coupling causes increased mitochon- OXPHOS involvement in atherogenesis. Athero- drial respiration but reduced phosphorylation with low sclerosis is the major cause of ischemic heart disease. ATP generation [34]. The excess metabolic turnover From the 1960s, Whereat emphasized that abnorm- produces heat instead of ATP, resulting in a steady alities of mitochondrial oxidative phosphorylation elevation of body temperature. Muscle fibers show might play an essential role in atherogenesis [42,43]. evidence of attempts to compensate for loose coupling He suggested that anoxia or carbon monoxide or “other in the form of aggregates of large mitochondria full of mitochondrial electron transport chain inhibitors that cristae [35], the primary location of OXPHOS contaminate our hazardous environment” could enzymes. Compensatory increase of mitochondrial initiate atherosclerosis [44]. synthesis may occur in the presence of other causes of Moreover, and mitochondrial dysfunction defective oxidative phosphorylation [36,37]. might help explain the cause of lipid accumulation in the atheromatous plaque. For instance, local arterial Damage and repair. 1. Reactive oxygen metabolites. hypoxia might increase endothelial permeability. This The reactive oxygen metabolites (ROM) generated would in part explain the lipid insudation theory that during mitochondrial respiration can damage increased permeability promotes mural insudation of membranes, DNA, and mitochondrial oxidative plasma lipid. Experiments show that reduced oxygen phosphorylation enzymes, causing a vicious cycle of tension or the presence of carbon monoxide accelerates declining mitochondrial function [38]. For instance, the development of atherosclerosis in the rabbit fed a in the cultured endothelial cell and the vascular smooth high fat diet. In humans, the increase in blood carbon muscle cell reactive species preferentially damage monoxide in tobacco smokers might be the link mtDNA compared with the transcriptionally inactive between tobacco smoke and atherosclerosis [44]. nuclear β-globin gene. The mitochondrial mRNA As an alternative to this insudation hypothesis it transcripts, protein synthesis, and ATP synthesis has been postulated that hypoxia enhances mural lipid decreased. Smooth muscle cells suffered less accumulation in atherosclerosis through fatty acid impairment than the endothelial cells [39]. synthesis by mitochondria in the aorta. Whereas Diseases of oxidative phosphorylation often occur normally fatty acid synthesis in the aorta is minimal, a together with impaired β-oxidation. Reduced flux block in the respiratory chain, for instance by hypoxia, through the respiratory chain increases the NADH/ impairs the oxidation of NADH and causes NADH NAD+ ratio. Beta-oxidation is inhibited and produces accumulation that initiates mitochondrial fatty acid secondary carnitine deficiency. Generation of reactive synthesis [44,45]. Molecular pathology of the mitochondrion 29

Involvement of defective oxidative phosphorylation instance, atherosclerotic aneurysms of the abdominal in atherogenesis is supported by experiments showing aorta of 13 patients contained on average 29 times more lack of mitochondrial ATPase and succinate PGE2 and almost 8 times more IL-6, compared to 16 dehydrogenase activity in cultured aortic vacuolated normal abdominal aortas [52]. smooth muscle cells from the atherosclerosis- IL-6 induces haptoglobin (Hp) and both contribute susceptible pigeon [46]. Furthermore, there is evidence to and angiogenesis [50]. Haptoglobin that reduced intimal mitochondrial energy generation binds hemoglobin [53] and serves as an important at lesion sites contributes to lesion formation in antioxidant. Its angiogenic activity was first susceptible compared with the resistant pigeon [45]. demonstrated in 1993 [54]. Both the in vitro Matrigel 2. Inflammation and mitochondrial dysfunction. model of angiogenesis and in vivo models showed that (a) Cyclooxygenase induction. Recent experimental purified haptoglobin stimulated angiogenesis [54]. It evidence, particularly using gene knockout animal was suggested that haptoglobin might be important models, not only corroborates the idea that dysfunction for angiogenesis as part of tissue repair in chronic of oxidative phosphorylation plays an important part inflammatory conditions [54]. A marked increase in in atherogenesis but also points to a close association the haptoglobin was detected in protein extracts from between inflammatory components in the athero- human aortic fibro-fatty lesions but not in aortic intima sclerotic lesion and mitochondrial dysfunction. In an from patients without atherosclerosis [55]. inflammatory environment oxidized lipids are Haptoglobin enhances cholesterol crystallization in the generated by the activity of lipoxygenases and bile [56], but a similar mechanism has not yet been cyclooxygenases. Mitochondria release radical oxygen demonstrated for the cholesterol crystal formation in species that deplete the cellular content of reduced the atherosclerotic lesion. glutathione. Oxidized fatty acids and oxidized forms (c) The need for cholic acid in atherogenic diets. High of cholesterol accumulate. These alterations are assoc- fat diets that are commonly used to induce athero- iated with apoptosis and necrosis as seen in the necrotic sclerotic lesions in animal models of atherogenesis core of the advanced atherosclerotic lesion [47]. contain cholic acid [57]. It induces nitric oxide (NO) The expression of cyclooxygenase-2 (COX-2) and synthesis in endothelial cells and reduces apoA-I [58]. other pro-inflammatory mediators has been demon- Dihydrocholic acid induces COX-2 [59]. Whereas strated in macrophages, smooth muscle cells, and high-fat, high-cholesterol diets without cholic acid endothelial cells of small vessels in the atherosclerotic resemble human diets more closely and elevate LDL plaque [48]. Others described finding an immuno- and HDL, they do not induce atherosclerosis in reactive 70-kDa COX-1 protein and a smaller, 50-kDa animals that are not genetically prone to develop COX-2 protein. The COX-2 isoform was located atherosclerosis. Addition of cholic acid elevates LDL together with macrophages primarily at the perimeter but lowers HDL and overcomes the resistance to the of the lipid core and the shoulder area of the atheroma development of atherosclerosis. Findings obtained in and secondarily in the microvascular endothelium of the mouse model of diet-induced atherosclerosis partly the lesion. By comparison, the normal artery only explain the reduction in the plasma levels of HDL. expresses the constitutive COX-1 isoform [49]. The addition of cholic acid to the diet induces the (b) Prostaglandin synthesis. COX-2 converts expression of apoA-I regulatory protein-1 and decreases arachidonic acid to prostaglandin-E2 (PGE2), which the expression of apoA-I [60]. induces interleukin-6 (IL-6) [50]. PGE2 inhibits (d) Inhibition of electron transport by interferon- cholesterol esterification in vitro, and might do the gamma. CD4+ and CD8+ T-cells in the atherosclerotic same in the arterial wall [51]. PGE2 extracted from lesion of the apoE null mouse secrete interferon-gamma lesions of rabbits rendered atherosclerotic by (INF-γ). Targeted disruption of the IFN-γ receptor in supplementing their diet with 1% cholesterol inhibited such mice reduces lesion size, cellularity, and lipid cholesterol esterification in vitro [51]. The levels of content and indicates that IFN-γ contributes to lesion PGE2 and IL-6 in atheromas vary greatly but are formation [61]. In vitro discoveries corroborate these significantly different from normal mural levels. For observations. IFN-γ induced the inducible nitric oxide 30 Annals of Clinical & Laboratory Science

(NO) synthase (iNOS) of the endotoxin-activated 3’UTR of the NDUFV1-mRNA that codes for a 51 J-774 macrophage cell. The NO inhibited the mito- kDa nuclear subunit of Complex I [64]. However, chondrial respiration of coincubated L-929 fibroblasts while plausible, it is presently not known whether this [62]. It competed with oxygen at Complex IV of direct INF-γ-link to a deficiency of mitochondrial cultured L-929 fibroblasts and reversibly inhibited oxidative phosphorylation operates in the athero- Complex IV. Others have shown that Trolox, a lipid- sclerotic lesion as well. soluble vitamin E analogue, but not ascorbate, can 3. Infectious agents. Chlamydia pneumoniae protect against NO-induced damage of cytochrome can damage mitochondria; they become oxidase, suggesting that the damage is mediated swollen and their cristae become fragmented [65]. through lipid peroxidation [63] (Figure 2). Gene Chlamidia, other bacteria and several types of viruses knockout and in vitro studies indicate that the nitric have repeatedly been detected in the human athero- oxide secreting, activated macrophage in the athero- sclerotic lesion, suggesting that some infectious agents sclerotic lesion might very likely inhibit respiration, initiate atherosclerosis [66]. Moreover, infection with not only of its own mitochondria, but also of cytomegalovirus (CMV) is significantly associated with mitochondria in other lesion cells, such as the smooth coronary heart disease, particularly in patients with muscle cell [62]. diabetes [67]. And in the New Zealand White rabbit A direct molecular connection between inflam- fed a 2% atherogenic diet the intravenous inoculation mation and Complex I deficiency has also been with the Herpesvirus type-4 (BHV-4) accelerated the proposed based upon the study of inflammatory development of atherosclerotic lesions [68]. However, myopathies. A complete antisense homology was experiments with germ-free apoE(-/-) mice showed that detected between the 5’UTR of the mRNA for the a bacterial or viral infection is not a sine qua non for INF-γ-inducible precursor protein (IP-30) and the the development of atherosclerosis [69]. 4. Observations in gene knockout and transgenic mice. (a) The apoE knockout mouse. The apoE(-/-) mouse develops massive atherosclerosis on a normal diet. Surprisingly, the presence of just a small amount of systemic apoE, less than 2% of the wild type, prevents lesion development. When transgenic apoE- knockout mice that had been engineered to express apoE strictly in the adrenal gland were fed an atherogenic diet, it was found that such low levels of systemic apoE blocked atherosclerotic lesion development in the aorta even in the presence of hypercholesterolemia [70]. Four weeks after bone marrow transplantation to generate macrophages expressing various forms of apoE in the murine apoE null mouse, the macrophages expressing murine apoE significantly reduced the size of atherosclerotic lesions Fig. 2. Links between inflammation and inhibition of and lowered the serum cholesterol level. However, the electron transport and proton-pumping Complexes I and macrophages expressing human apoE3-Leiden or IV in atherogenesis. The illustration is based upon in vitro apoE2 did not curtail lesion size [71]. γ findings that interferon-gamma (INF- ) released by CD4 Infection with the murine gamma-herpesvirus-68 and CD8 cells induces the inducible nitric oxide synthase (MHV-68) accelerated atheroma formation in infected (iNOS) in the activated macrophage (Mø]. Nitric oxide inhibits Complex IV. Secondly, INF-γ inhibits expression apoE(-/-) mice compared with control mice. [72]. By of the nuclear gene NDUFV1 that codes for a subunit of contrast, addition of oral Q10 to a high-fat diet given Complex I. MIM: mitochondrial inner membrane; SMC: to uninfected apoE null mice significantly reduced the smooth muscle cell. size of atherosclerotic lesions in the aorta [73]. Molecular pathology of the mitochondrion 31

(b) The LDL-receptor knockout mouse. The LDL- The LDL-receptor down-regulation can also be receptor knockout mouse, LDLR(-/-), develops bypassed in vitro by covalent linkage of N,N-dimethyl- atherosclerotic lesions of the thoracic and abdominal 1,3-propanediamine (DMPA) to LDL, leading to vast aorta after 12 weeks on a diet not supplemented with uptake and extensive accumulation of intracellular lipid cholic acid. Two groups of mice were fed either a high- inclusions containing liquid crystals of cholesteryl fat diet or a high-fat diet supplemented with sodium esters. These in vitro features resemble the alterations cholate. Similar lesions developed in both groups that are found in smooth muscle cells in vivo during demonstrating that the receptor defect overcomes the atherogenesis [80]. HIPDM (N-N-N’-trimethyl-N’- resistance to develop atherosclerosis on a high-fat diet (2-hydroxy-3-methyl-5-iodobenzyl)-1,3 propane- not supplemented with cholic acid [74]. diamine) is structurally similar to DMPA. It accumul- Overexpression of LPL can protect against ated preferentially in mitochondria when given atherosclerosis in the LDL-receptor (LDLR) deficient intravenously to rabbits, and its distribution was similar mouse. At the end of an 8-week atherogenic diet the to succinate cytochrome c reductase. It remained in LDLR(-/-) mouse with transgenic overexpression of mitochondria for at least five hours after injection [81]. human LPL had 18-fold smaller lesion areas, compared (b) Intracellular defect in fuel transport and delivery with the LDLR(-/-) mouse not possessing the LPL to mitochondria. A study of human atherosclerotic transgene. It was suggested that the LPL-induced plaques material removed percutaneously showed many reduction in plasma levels of remnant lipoproteins smooth muscle cells, mainly of the intermediate caused the reduction in lesion area in the mice that phenotype, having abundant perinuclear fat and overexpressed LPL [75]. glycogen deposits and many mitochondria [36]. The In ovariectomized LDLR(-/-) mice, aortic lesion increase in the number of mitochondria in the size was reduced by physiologic amounts of exogenous atherosclerotic lesion indicates enhanced synthesis in 17β-estradiol. The effect was independent of an attempt to compensate for dysfunction of the alterations of the cholesterol concentration in the mitochondrial bioenergetics system. The perinuclear plasma [75]. lipid accumulation is similar to that in Reye’s syndrome. (c) Lipoprotein lipase overexpression. Observations There is evidence suggesting that the mitochondrial in the muscle-specific LPL transgenic mouse suggest permeability transition (MPT) is involved in that the lipoprotein lipase activity might be a rate- mitochondrial injury of the liver in this rare disorder. limiting step in the triglyceride-derived supply of free It is strongly associated with viral infection and fatty acids to mitochondria and that LPL expression concomitant aspirin ingestion and appears primarily participates in the regulation of the biogenesis of [82] but not always [83] in childhood. mitochondria. The overexpression of LPL in cardiac 6. Defective oxidative phosphorylation in and skeletal muscle increased the uptake of free fatty atherogenesis. (a) Inherited structural defects. Direct acids and induced muscle fiber degeneration, glycogen evidence of a deficiency of respiratory enzymes in storage, and marked mitochondrial proliferation [77]. atherogenesis was provided by two cases of metabolic 5. Defective fuel supply. (a) Defect in the cellular disease with premature atherosclerosis considered of uptake of fuel. As noted above, the apoE knockout mitochondrial origin. The premature atherosclerosis mouse develops severe atherosclerosis. LDL from aortic was diagnosed in two brothers whose parents and two atherosclerotic lesions (A-LDL) is chemically modified sisters were free of symptoms. The brothers, who died and is not taken up as much as normal LDL by rabbit prematurely in their third decade of life, also suffered aortic smooth muscle cells (SMC) in vitro. Both from diabetes mellitus, sensorineural deafness, lipoproteins down-regulate the cell surface LDL photomyoclonic epilepsy, and progressive renal failure. receptor and both failed to induce cellular cholesterol- The mitochondrial abnormalities consisted of partial ester accumulation in the cell [78]. However, deficiencies of Complexes III and IV. The enzyme incubation of human arterial SMC with human defect could be ascertained in the kidney and in fibro- chylomicron remnants increases cell cholesterol and blasts, but not in muscle. Whether the mutation was suppresses LDL-receptor activity [79]. located in the mtDNA or the nDNA coding for 32 Annals of Clinical & Laboratory Science mitochondrial enzyme subunits was not resolved [84]. demonstrated using chloroquine, a Q10 analog. (b) Acquired structural defects. Somatic mtDNA Chloroquine reversibly inhibits electron transport and damage accumulates with age, degrades oxidative cell growth [91]. In an in vitro model for lipid accum- phosphorylation and accelerates aging. In the normal ulation in atherosclerosis, smooth muscle cells from human heart the ‘common’ 4,977 nucleotide pair (nt) pig aortas were incubated with LDL, chloroquine, or deletion (mtDNA4,977) and the mtDNA7,436 and both. LDL supplemented cells showed normal mtDNA10,422 deletions accumulate with age above morphology save for a few cells containing large lipid 40. In atherosclerosis of the coronary arteries this droplets. By comparison, chloroquine was toxic to somatic damage is significantly accelerated. In human the cells, which developed large autophagic vacuoles. hearts with coronary artery disease (CHD), the LDL and chloroquine together induced autophagic ‘common’ deletion is up to over 200 times more vacuoles, large lipid droplets, and a marked increase in frequent than in age-matched control hearts free of esterified cholesterol [92]. Treating the remnant cells CHD [85]. with chloroquine (30 mM) increased the content of The ‘common’ deletion abolishes the mitochondrial cholesterol by 90% and cholesterol ester by 370% [79]. synthesis of subunits for Complexes I, IV, and V. (e) Cellular regulation of intracellular cholesterol Significantly higher levels of this deletion were detected synthesis. It has been postulated that the protective by the polymerase chain reaction (PCR) in subjects effect of estrogen against heart disease is due to its aged 73-95 compared with subjects aged 60-72 years tissue-specific regulation of 3-hydroxy-3-methyl- old in mtDNA extracted from smooth muscle cells of glutaryl coenzyme A (HMG-CoA) reductase, the main aortic atherosclerotic lesions from 18 surgical and 9 regulator of the isoprenoid metabolic pathway. It cases. These findings support the theory that converts 3-hydroxy-3-methylglutaryl coenzyme A defective oxidative phosphorylation contributes to the (HMG CoA) to mevalonate. Its promoter region degenerative cell phenotype and atherosclerosis during harbors a sequence element that differs from the aging [86]. consensus sequence of the estrogen-responsive element (c) Reperfusion injury. Cardiac mitochondria isolated (ERE) by only one mismatch in each half of the after reperfusion are structurally abnormal, carry large palindrome. It is unresponsive to estrogen in the liver amounts of ionized calcium, and generate a dispro- but responds to estrogen in other tissues that require portionate quantity of oxygen free radicals that cause enhanced cell proliferation [93]. Lipoprotein-deficient deletions of mtDNA. The oxidative phosphorylation serum from women using oral contraceptives induced is irreversibly damaged [87]. Myocardial recovery after HMG-CoA reductase in the cultured human aortic ischemia and reperfusion of isolated perfused hearts smooth muscle cell [94]. ATP inhibits HMG-CoA from rats was not improved by prior Q10 supplementa- reductase [95,96]. tion and myocardial Q10 content was unaffected [88]. 7. Speculations; filling gaps in present knowledge. In the dog model of reperfusion, the PGE2 level in This review of atherosclerosis analyzes and correlates vivo in the great cardiac vein and ex vivo in heart present experimental evidence of defective oxidative mitochondria changed significantly after 20 minutes phosphorylation in its etiology. A few suggestions are of left artery occlusion. Pretreatment with indo- introduced here to fill the gaps in our understanding, methacin inhibited the PGE2 increase [89] suggesting as present experimental data are insufficient to establish cyclooxygenase-2 induction or activation by the a unified theory of the pathophysiology involved. reperfusion. When isolated mitochondria were exposed (a) Reduced binding of liposomes to mitochondria to PGE2 in the presence of ionized calcium in vitro, reduces fuel transfer. The apoE null mouse develops proton conductivity increased, leading to uncoupling, atherosclerotic lesions even when fed a diet free of inhibition of respiration, and lowering of ATP synthesis cholic acid supplementation. The common explan- [90]. ation is that the lack of apoE inhibits removal of lipo- (d) Inhibition of the electron transport chain results proteins by the liver, leading to accumulation of athero- in intracellular lipid accumulation. The essential role genic lipoproteins in the circulation. ApoE binds to of Q10 in the electron transport system has been the cellular surface low-density lipoprotein (LDL) Molecular pathology of the mitochondrion 33 receptor, also known as the apo B,E(LDL) receptor, (b) Lack of LPL. Furthermore, one might speculate and the LDL-related protein (LRP). In addition, it that intracellular LPL induces increased release of fatty binds to a 59-kDA intracellular apoE binding protein acids from mitochondrial-bound liposomes for identified to contain the α- and β-subunits of oxidative phosphorylation and thus protects intimal F1-ATPase. It was detected in the canine and human cells and reduces the lesion areas in the LDL-receptor liver cell [97]. null mouse. A defect in LPL synthesis would therefore As an alternative explanation of intracellular lipid not only interfere with intravascular but also with accumulation, I suggest that the intracellular lipid- intracellular lipolysis (Figure 3). transport system utilizes liposomes that bind via their (c) Defective oxidative phosphorylation. Reduced ATP surface apoE to associate with mitochondria. This idea production due to defective OXPHOS reduces is derived from the above reports of intracellular apoE- inhibition of HMG-CoA-reductase activity, and binding proteins that are imported into the mitochon- increases synthesis of Q10 to enhance OXPHOS, but drion. A lack of liposome binding via apoE to the simultaneously increases cholesterol synthesis. mitochondrial protein in the apoE-null mouse would The gene for COX-2 is located at chromosome lead to a defect in the lipid fuel for mitochondrial region 1q25, the same region as the gene for cytosolic energy conversion and to intracellular lipid accum- phospholipase (cPLA2), and it has been suggested that ulation. Similarly, in humans, such reduced binding the two genes are co-regulated [98]. cPLA synthesizes by apoproteins like apoε4 might contribute not only arachidonic acid from membrane phospholipids. to atherogenesis, but also to other diseases such as Arachidonic acid selectively inhibits Complexes I and Alzheimer’s disease. III and significantly elevates mitochondrial hydrogen

Fig. 3. Illustration showing evidence-based and hypothetical role of dysfunctional oxidative phosphorylation (OXPHOS) in atherogenesis. A combination of inflammation (COX-2 induction) and dysfunction of OXPHOS causes the atherosclerotic lesion. The latter induces HMG-CoA reductase and increases the synthesis of Q10 as well as cholesterol as they share the same pathway via mevalonate and farnesyl. Hypercholesterolemic diets used to induce experimental atherosclerosis contain bile acids, which inhibit OXPHOS and induce COX-2. Abbreviations: LP: lipoprotein; LDLR: low-density lipoprotein receptor; LRP: low-density lipoprotein receptor related protein; LPL: lipoprotein lipase; CHOL: cholesterol; FA: fatty acid; apoE: apolipoprotein E; SMC: smooth muscle cell; A: atherosclerotic artery, cross section view; COX-2: cyclooxygenase-2; PGE2: prostaglandin E2; IL-6: interleukin-6; Hp: haptoglobin; CoQ10: coenzyme Q10 (ubiquinone), CE: cholesteryl ester. For further explanation see text. 34 Annals of Clinical & Laboratory Science peroxide production. Unsaturated acid inhibits more with deafness [101]. Diabetes causes impairment of than saturated acid [99]. These findings suggest uptake into cells. It is an independent risk another link between inflammation and OXPHOS. factor for atherosclerosis [102]. The pancreatic β-cell The co-localization of HIPDM with succinate depleted of mtDNA does not secrete insulin [103]. cytochrome c reductase suggests that the enzyme might Beta cells with mtDNA mutations show a defective be inhibited by HIPDM, causing accumulation of mitochondrial inner membrane potential and induce intracellular lipids, reduced ATP synthesis, less diabetes. Insulin resistance is closely related to the inhibition of HMG-CoA reductase, increased Q10 regulation by uncoupling proteins and other energy synthesis and simultaneous increase in cellular regulators [103]. The high-glucose environment cholesterol synthesis. induces glycation of proteins. The concurrent ROS- (d) Combined defects. The detrimental effects of generation induces apoptosis in vascular cells involved reduced fuel supply and inhibition of oxidative in complications of diabetes mellitus [103]. phosphorylation on cellular energy metabolism is Maternally inherited diabetes and deafness demonstrated by Reye’s syndrome. Aspirin has been (MIDD) is a rare disease found only in 1% to 2% of shown to inhibit β-oxidation and thus the fuel supply, individuals with diabetes. Enzyme activities of less than and certain viruses induce the MPT. Additional five percent of the tolerance levels of Complexes I, I+III, damage could be induced by salicylate, a metabolite and IV have been detected in skeletal muscle biopsies. of aspirin. Applying the same reasoning to athero- Modification of diet and increased exercise has provided genesis, it appears reasonable to assume that the only temporary improvement [104]. inhibition of the availability of lipid fuel to mitochon- In the rat model of diabetes induced by strepto- dria combined with drug- or virus-induced direct zotocin (STZ) the mitochondrial oxidative phosphory- inhibition of oxidative phosphorylation would lation is significantly reduced. However, the ATP significantly reduce ATP generation and be highly generation can be completely restored by physical detrimental for cell survival. For instance, as noted training even if the plasma glucose or insulin levels above, the Herpesvirus accelerates experimental athero- remain essentially unaltered [105]. Others have sclerosis. A way for estrogen to protect intimal cells reported that endurance exercise training increased would be the increased synthesis of Q10 by stimulation oxidative capacity of the muscle and doubled the of HMG-CoA-reductase. The findings that ovariec- number of mitochondria in rat muscle [106]. Reduced tomized rabbits given estrogen do not develop oxygen consumption per mitochondrion might lead atherosclerosis even in the presence of a high blood to less oxygen radical generation per mitochondrion cholesterol level suggest that the atherogenic defect is with less damage to individual mitochondria [107]. mural, and that local cellular regulation is most IL-1β is an acknowledged mediator of dysfunction important in atherogenesis. This is also supported by of the pancreatic β-cell in Type I diabetes mellitus. the results showing that when Q10 is blocked by Differential-mRNA display reveals that IL-1β induces chloroquine, the low ATP level induces an increase in ANT1 expression in cultured, purified rat β-cells receptor uptake. [108], suggesting an altered ANT isoform expression. 8. Cardiomyopathy. Several mtDNA mutations There is in vitro evidence that sulphonylureas might and reduced respiratory enzymes activities have been impair mitochondrial function. For instance, tolbut- associated with hypertrophic cardiomyopathy. For amide can depolarize the mitochondrial potential of instance, deficiencies in respiratory chain enzyme the cultured pancreatic β-cell [109]. activities were detected in half of a series of 32 endocardial biopsies. The activity of either Complex Gastrointestinal disease. 1. The “topical” effect in ulcer I, IV, or both, was significantly reduced relative to the formation. (a) Nonsteroidal anti-inflammatory drugs: combined activities of Complexes II and III [100]. Local inference with mucosal mitochondrial oxidative phosphorylation by nonsteroidal anti-inflammatory Diabetes. Mitochondrial DNA with large deletions drugs (NSAIDs) is pivotal in the development of has been detected in patients with diabetes associated gastrointestinal ulcers in patients treated with them. Molecular pathology of the mitochondrion 35

Indeed it has been postulated that this “topical” phase glycolytic substrate, provided partial protection against is a sine qua non of NSAID-induced gastrointestinal the ATP depletion and cell injury induced by these damage [110]. compounds [117]. Like bile acids, nonsteroidal anti-inflammatory Direct evidence that uncoupling of oxidative drugs can inhibit oxidative phosphorylation [111]. phosphorylation is important in the pathogenesis of Lipophilic NSAIDs are readily transported through the NSAID-induced gastrointestinal side effects was cell wall and are enriched within the mitochondrion obtained from studies of intestinal damage in rats [112]. Whereas the anti-inflammatory effects of caused by parenteral administration of either aspirin, NSAIDs are due to inhibition of COX-2, the the uncoupling agent 2,4-dinitrophenol, or indo- concurrent inhibition of COX-1 by non-selective methacin. Aspirin given orally inhibited intestinal NSAIDs and their interference with mitochondrial mucosal cyclooxygenase without causing a topical oxidative phosphorylation can cause serious gastro- effect: it increased mucosal permeability and reduced intestinal side effects. NSAIDs such as indomethacin mucosal prostanoid levels but did not alter are very effective analgesic and antiphlogistic agents mitochondrial morphology. In these experiments, and are widely used, resulting in a large number of aspirin caused neither significant inflammation nor patients that experience side effects of the treatment. ulcer formation. However, the mitochondrial For instance, it has been estimated that NSAID- uncoupler 2,4-dinitrophenol increased intestinal induced hemorrhage and ulcerations cause up to permeability, but had no effect on intestinal prostanoid 20,000 deaths yearly in the United States [17]. levels. In contrast, the two agents administered Indomethacin uncouples oxidative phosphorylation together induced alterations similar to those induced at micromolar concentrations and inhibits respiration by oral indomethacin administration such as altered at higher concentration in vitro [113]. Oral mitochondrial morphology, increased permeability, administration of indomethacin in the rat significantly decreased prostanoid levels and the formation of lowered ex vivo jejunal ATP and α-tocopherol levels intestinal ulcers [118]. [114]. Electron microscopic examination of the Five hours after application directly to the human mucosa revealed that it caused dose-dependent gastric mucosa, aspirin induces dilatation of mitochondrial changes comparable to those inducible mitochondria and the , in vivo by the classical mitochondrial uncoupler rupturing of apical membranes, intercellular edema, dinitrophenol [113]. Other NSAIDs such as nime- and widening of capillary fenestrae [119]. These sulide, meloxicam, and piroxicam also uncouple findings resulted from a controlled clinical trial mitochondria and stimulate respiration in vitro [115]. involving 5 healthy volunteers as probands and 5 other They stimulate basal and uncoupled respiration in healthy subjects as controls. All volunteers were free of mitochondria incubated in the presence of either Helicobacter pylori (H. pylori) infection (a significant glutamate plus malate or succinate [115]. Diclofenac, point, vide infra). It has also been suggested that aspirin but not naproxen, also slightly inhibits ATPase activity. at relative concentrations similar to human antipyretic Furthermore, nimesulide and diclofenac, but not and anti-inflammatory pharmacological doses can naproxen, reduce ATP synthesis by blocking the activity uncouple oxidative phosphorylation of rat of the adenine nucleotide translocase [115]. mitochondria in vivo and in vitro [120]. However, Diphenylamine is a shared structure of several these in vivo experiments might not have differentiated NSAIDs that are able to uncouple mitochondrial between the effect of aspirin and its salicylate oxidative phosphorylation. For instance, diphenyl- metabolite. amine, mefenamic acid, and diclofenac produce Importantly, even if an agent itself does not affect mitochondrial swelling of rat liver mitochondria oxidative phosphorylation, one of its metabolites might obtained from freshly isolated and decrease do so. As an example, salicylate, a main metabolite of cellular ATP content, mainly through uncoupling of aspirin, but not aspirin itself, uncouples oxidative the mitochondrial oxidative phosphorylation [116]. phosphorylation. A close correlation between the An important finding was that fructose, an effective uncoupling activity of salicylate and its congeners and 36 Annals of Clinical & Laboratory Science their anti-inflammatory potency had been noted almost synthesis via specific inhibition of Complex I of the forty years ago [112]. Acetylsalicylic acid in the low respiratory chain of cultured cells, and in the whole millimolar range both uncoupled and inhibited heart nabumetone reduced the oxygen uptake [115, oxidative phosphorylation of rat renal cortex 113]. Moreover, cultured cells and the whole heart mitochondria in vitro; in comparison, the NSAID exposed to nabumetone showed a reduction in oxygen dipyrone only uncoupled OXPHOS [121]. Other uptake indicating in vivo inhibition of the respiratory investigators reported that salicylate induced the chain [115]. Nonetheless, clinical studies indicate that mitochondrial permeability transition (MPT), depleted the COX-2 selective inhibitor celecoxib, which does the mitochondrial inner membrane potential and not affect oxidative phosphorylation, and nabumetone uncoupled oxidative phosphorylation [27]. are somewhat similar in their rate of ulcer induction. Different NSAIDs differ in their effect on Both drugs show about a 4-fold reduction in ulcer mitochondria, and their effects on mitochondria may complications vs comparator NSAIDs. [124,125]. be concentration dependent. A number of NSAIDs (d) The effect of DMSO. In many comparison animal and acidic prodrugs stimulate mitochondrial respir- studies of the gastrointestinal effects of various ation in vitro and uncouple mitochondria. NSAIDs, eg indomethacin versus celecoxib, the drugs Modification or removal of the ionizable group can are diluted in dimethyl sulphoxide (DMSO). When eliminate the adverse effect on mitochondria. For DMSO is used as a vehicle for suspension of lipophilic instance, dimeroflurbiprofen, a modified flurbiprofen, NSAIDs in animal or clinical studies it might alter the non-acidic prodrugs such as nabumetone, and non- effect of the drugs on oxidative phosphorylation. An acidic selective COX-2 inhibitors do not cause in vitro important, sometimes overlooked, variable in studies uncoupling [122]. of NSAIDs is the effect of DMSO itself. As an (b) Helicobacter pylori. NSAIDs that do not induce example, isolated rat kidney mitochondria exposed in the “topical” phase of gastrointestinal damage, such as vitro to cyclosporin A with and without DMSO were the selective COX-2 inhibitors celecoxib and rofecoxib, minimally affected. However, when exposed to DMSO cause fewer gastric ulcers than indomethacin in animal alone there was an increase of about 20% in experiments and are better tolerated in clinical trials spontaneous ATP generation [126]. The NSAID [123]. However, infection with the gastrophilic Gram- imidazole causes hyperpolarization of the mitochon- negative H. pylori might increase the risk of ulcer drial membrane in cultured mouse erythroleukemia formation in patients receiving such NSAID therapy cells exposed to DMSO as opposed to membrane because the vacuolating cytotoxin (VacA) released by depolarization observed with DMSO alone [127]. In the bacterium might induce the “topical” effect. addition, the vehicle can affect different drugs Support for this assumption has evolved from in vitro differently [128]. It has been suggested that DMSO observations: vacuolating cytotoxin prepared from H. itself may be an anti-inflammatory agent [129]. DMS, pylori decreases ATP levels and increases the number a known metabolite of DMSO, but not DMSO, of cultured gastric cells in the G0/G1 phase. The produces mitochondrial uncoupling in vitro [130]. It cytotoxin decreases the potential ∆Ψm across the might explain the observation that DMSO mitochondrial inner membranes of the cultured gastric administration to rats induces structural alterations in AZ-521 epithelial cell [31]. mitochondrial membranes leading to enhancement of (c) Non-acidic and COX-2 selective NSAIDs. One cytochrome oxidase activity in liver mitochondria might presume that non-acidic NSAIDs such as [131]. nabumetone and NSAIDs that have no enterohepatic 2. Restoration of apoptosis in neoplasia. The early circulation do not induce a ‘topical” phase. Never- observation that lipophilic NSAIDs are readily trans- theless, it has been reported that nabumetone can ported through the cell wall and are enriched within inhibit mitochondrial respiration. It limited the the mitochondrion [112] has attracted much recent mitochondrial inner membrane potential and reduced attention. This has happened because acidic NSAIDs ATP synthesis via specific inhibition of Complex I of can initiate apoptosis in many tumors, for instance the respiratory chain [115]. Nabumetone reduced ATP colorectal . The fact that some NSAIDs Molecular pathology of the mitochondrion 37 exhibit significant anti-tumor effects was first detected in ATPase activity in the rat liver mitochondrion [137]. in epidemiological studies and later verified in animal In addition, it has been found that chronic ingestion studies and in clinical trials. A major part of this effect of ethanol significantly reduced the concentration of is due to the NSAID-induced inhibition of cyclo- two mtDNA-encoded polypeptides, subunits 8 and oxygenase-2 (COX-2) in the tumor, and selective 6, of the F0F1-ATPase in the rat liver. In contrast, COX-2 inhibitors have been shown to prevent or ethanol consumption had neither an effect on the inhibit the growth of certain tumors [123]. In compar- nuclear encoded subunits of F0F1-ATPase nor on the ison, a significant part of tumor growth inhibition by nuclear-encoded adenine nucleotide transporter [138]. non-selective, acidic NSAIDs is due to the NSAID- It has been suggested that a myocardial metabolite induced depolarization of the mitochondrial inner of ethanol, fatty acid ethyl ester (FAEE), might be a membrane potential that brings about the MPT and link between chronic ethanol abuse and myocardial restores apoptosis [50]. dysfunction. In the rabbit, FAEE binds to myocardial mitochondria in vivo and in vitro and can be hydro- Ethanol-induced disease. Ethanol depresses the lyzed to fatty acid, an uncoupler of oxidative phosphor- oxidative phosphorylation of hepatic mitochondria ylation [139]. from rats fed ethanol chronically [132]. Such mito- Ethanol at a concentration of 1 mM and 10 mM chondria display a reduced capacity to incorporate 35S- lightly reduces cell viability of cultured rat hepatocytes methionine into mitochondrial polypeptide gene and increases the combined ROS generation at products in vitro. The ethanol exposure reduces the Complexes I and III about 70% and 150% respectively steady-state concentration of every mitochondrial gene [140]. Whereas ethanol consumption significantly product. However, below a threshold, ethanol increases ROS generation by the rat hepatocytes in such enhances mitochondrial DNA replication, for instance in vitro studies, the loss of viability is mostly in the ethanol-treated embryo [133]. correlated with a decrease in the cellular ATP level The rat liver mitochondrion exposed to ethanol in [141]. Moreover, the amount of dietary fat (high-fat vitro exhibits malfunction of the transporter of or low-fat diet) has no significant effect on these glutathione (GSH). As a result, the mitochondrial ethanol-induced alterations [141]. Remarkably, matrix is depleted of GSH that normally metabolizes supplementation with fructose [140] or pretreatment hydrogen peroxide. The mitochondrion becomes more with the alcohol dehydrogenase inhibitor 4-methy- susceptible to alcohol-induced oxidative stress. lpyrazole (4-MP) [142] prevented in vitro ethanol- However, the effect can be prevented by supplement- induced cytotoxicity. In a rat model of the fetal alcohol ation with S-adenosyl-L-methionine [134]. syndrome, ethanol exposure in utero reduces perinatal Alcoholics may show abnormalities of mitochon- activities of Complexes II and IV and depresses dria even after cessation of ethanol intake [135]. The respiration of mitochondria from fetal hearts [143]. investigation of oxidative phosphorylation of mice that were fed ethanol might suggest an explanation. The Neurological disorders. 1. Primary and secondary studies revealed that the GSH levels were reduced and defects of OXPHOS. Recent evidence suggests that the extent of oxidation of mitochondria DNA was defective oxidative phosphorylation contributes to significantly increased. These alterations combined neurodegenerative disorders. Primary defects in the with the lack of an effective mtDNA repair system electron transport chain have been detected in might result in permanent damage to the mitochondria Alzheimer’s disease (AD) and Parkinson’s disease (PD). of alcoholic subjects [135]. In comparison, in Friedreich’s ataxia and Huntington’s Mitochondria from rats fed ethanol chronically disease, mutations of the nuclear DNA, probably show a reduction of total phospholipids (except for induced by free radicals, result in low levels of aconitase cardiolipin) in the inner membrane, alteration in the that cause secondary defects in oxidative phosphory- cytochrome content, and a decline of the ability to lation. Lack of ATP that lowers the threshold to produce ATP [136]. It was also demonstrated that undergo apoptosis may be a central mechanism in the chronic ethanol consumption caused a marked decrease pathogenesis of these neurodegenerative diseases [144]. 38 Annals of Clinical & Laboratory Science

2. Alzheimer’s disease. (a) The effect of aging and suggesting that βAPP is an important factor in the oxidative damage. Until recently the association of any pathogenesis of the two diseases [151]. mitochondrial defect with the etiology and 3. Parkinson’s disease. Mitochondrial DNA with pathogenesis of Alzheimer’s disease had been doubtful large 5kb deletions, eliminating genes encoding protein [145]. However, a few reports suggest that oxidative subunits essential for mitochondrial ATP synthesis, stress and mitochondrial dysfunction play a significant have been found in the striatum of patients with role in the pathogenesis [146]. It is probable that the Parkinson’s disease. However, such deletions were also age-related impairment of mitochondrial function found in aged subjects without the disease [152]. It induced by oxygen reactive metabolites might has been suggested that the significant increase of predispose or cause diseases such as Alzheimer’s disease Complex IV defects found in neurons of the substantia [147]. Importantly, it has been demonstrated that nigra are most likely due to accelerated aging [153]. brains from AD-patients show more nicking and Reduced activity of Complex I has been implicated in fragmentation of both the nuclear and mitochondrial both idiopathic Parkinson’s disease and Parkinsonism DNA. In addition, the amount of mtDNA was induced by the mitochondrial permeability transition reduced and 8-oxo-7,8-dihydro-2'-deoxyguanosine [154]. In vitro, the apoptosis-inducing neurotoxin N- (8-OH-dG) was detected by immunostaining methyl-4-phenylpyridinium (MPP+) inhibits Complex indicating oxidative damage. These alterations might I, depletes ATP, and induces release of cytochrome c predispose to further neuronal damage by exposure to by opening the membrane pores [155]. other endogenous or environmental factors [147]. Reduced availability of coenzyme Q10 may also (b) The effect of apoE4. The role of mitochondrial play a role in the etiology of Parkinson’s disease. In dysfunction might depend on the presence of apoE4 the mouse model, oral therapy with Q10 protected [148]. The apoε4 allele is associated with increased against the detrimental effects of PMTP on the H2O2-induced lipid peroxidation in the frontal cortex dopaminergic system of the substantia nigra [156]. of patients with Alzheimer’s disease [148]. Clinically, the presence of the ε4 allele of the apoE gene increases Neoplasia. There is evidence that somatic mutations the risk of dementia on average 5-fold and is of the mitochondrial genome may be involved in the independent of its effect on plasma lipids and etiology of cancer and that aggregation of non-somatic atherogenesis [149]. mutants may play a role in their progression. An In AD patients who carried the ε4 allele the clinical analysis using 2-dimensional gene scanning of mtDNA dementia rating (CDR) score correlated with from 21 papillary carcinomas of the thyroid gland diminished activity of the mitochondrial enzyme α- revealed 3 different somatic mutations in 5 of the ketoglutarate dehydrogenase. By contrast, in AD tumors, which mainly affected genes encoding enzyme patients without the ε4 allele the CDR correlated Complex I of the respiratory chain [157]. significantly better with the densities of neuritic plaques A reduced activity of Complex IV and V, the latter β and tangles [150]. Reduced levels of pyruvate dehydro- due to a decrease of the F1- moiety, was detected in genase and cytochrome oxidase have also been detected mitochondria from hepatocellular carcinoma [158]. in AD brain tissue [146]. The F0 moiety of the F0F1-ATPase of the rat liver (c) Amyloid precursor protein. Transfection of the mitochondrion contains a distinct binding site for gene for amyloid precursor protein (βAPP) into diethylstilbestrol (DES), and at low concentrations, it cultured normal human muscle fibers caused is a potent F0F1-ATPase inhibitor [159]. DES is overexpression of the protein and induced structural associated with the development of clear cell abnormalities of mitochondria and reduced adenocarcinoma of the vagina and cervix in women cytochrome-c oxidase activity. Abnormal accumulation exposed to it in utero. of βAPP and similar mitochondrial abnormalities with Neoplastic transformation can be associated with reduced activity of this enzyme complex have been markedly altered expression of both the nuclear and observed in the brain of patients with Alzheimer’s mitochondrial DNA encoded oxidative phosphoryl- disease and in patients with inclusion-body myositis ation genes [160]. For instance, an analysis of human Molecular pathology of the mitochondrion 39 diploid fibroblasts and their SV 40-transformed It has been suggested that fragments of counterparts revealed that the mtDNA number mitochondrial DNA released during mtDNA damage declined. However, the mRNA levels for the mtDNA- and incorporated into nDNA might cause cancer encoded 12 S rRNAs, ND2, ATPase6+8, COIII, [167]. Recent experimental evidence supports this ND5+6, and cytochrome b (CYTB) genes and the hypothesis: it was demonstrated that in the mouse and mRNAs for the nuclear-encoded ATP-synthase-β were rat mitochondrial-DNA-like inserts were much more increased. In addition, the adenine nucleotide abundant in tumors than in normal tissue [168]. translocator (ANT) isoform 1 and 2 genes were markedly induced. A different study found that the Molecular Pathology ATPase activity in mitochondrial particles from Zajdela hepatoma and Yoshida sarcoma was substantially lower Mitochondrial synthesis and turnover. 1. Nuclear than the control mitochondrial particles isolated from DNA. The biosynthesis of mitochondria is controlled rat heart and rat liver [161]. Tumor mitochondrial by the nucleus. Nuclear DNA codes for most particles contained 2 to 3 times more ATPase inhibitor mitochondrial proteins needed for the synthesis of than particles from control mitochondria. mitochondria and encodes most of the estimated 1000 A number of findings support a role for oxidative proteins required for proper OXPHOS function [169] stress and defective oxidative phosphorylation in (Figure 4). Remarkably, mitochondria in the ρ0-cell . For instance, biochemical alterations lack mtDNA but the cell still synthesizes mitochondria in biopsy tissue from 59 patients with gastric cancer in such cells, showing that nuclear DNA alone controls were compared with tumor-free, adjacent mucosa. All mitochondrial synthesis. tumors exhibited a significant impairment of function The nuclear genes that are phylogenetically of as evidenced by reduction in tetrazolium dye staining mitochondrial origin code for the many proteins that and of the antioxidant enzyme superoxide dismutase are imported into the mitochondria. For instance, the β (SOD), and glutathione S-transferase (GST) consistent import of the F1- subunit of the F1 moiety of F0F1- with oxidative stress. Positive staining with H. pylori ATPase requires both an intact membrane potential antibody significantly decreased the reduction in and ATP [170]. Nuclear coded proteins are imported tetrazolium staining [162]. into mitochondria typically after proteolytic elimin- Gastric cancer is associated with defective ation of their leader peptides and require an intact mitochondrial function in the tumor and the adjacent mitochondrial membrane potential and ATP [171]. tumor-free mucosa [162] and H. pylori infection might 2. Regulation and coordination of mitochondrial be involved in the pathogenesis of gastric cancer: 10% biosynthesis. By stimulating mitochondrial biogenesis, to 20% of infected cases develop ulcers and many human tissues seek to counteract defective oxidative develop atrophic gastritis [163]. phosphorylation, which might be caused by a mtDNA Mitochondrial dysfunction of Complexes I and IV mutation [172]. The exact molecular mechanism that has been observed in HIV-1-negative children whose regulates the coordinated mitochondrial and nuclear mothers had been administered a prophylactic gene expression for the complete synthesis of combination of the nucleoside analogue zidovudine mitochondria is uncertain [173]. Some evidence and lamivudine or zidovudine alone during pregnancy suggests that the control region of the F0F1-ATPase to prevent mother-to-child HIV-1 transmission [164]. genes coordinate the expression of the 2 genomes It has been suggested that AZT-induced oxidative depending on the energy demands of the cells, damage in nuclear DNA of fetal tissues might be the especially in muscle [174]. reason why it can be a perinatal carcinogen. For Experiments using the transgenic fruit fly example, treatment of CD-1 Swiss pregnant mice and Drosophila megaloblaster have provided evidence that the pregnant patas monkey (Erythrocebus patas) with the ubiquitous DNA binding transcription factor Sp1 AZT induces a significant increase in 8-oxo-2'- regulates the expression of nuclear genes involved in deoxyguanosine (8-oxo-dG) in fetal tissues such as the mitochondrial biogenesis [175]. In these experiments, liver and kidney [165,166]. promoter regions for the mitochondria transcription 40 Annals of Clinical & Laboratory Science

Fig. 4. The synthesis of mitochondria is controlled by the nucleus. An individual mitochondrion may have up to ten copies of mtDNA in its matrix compartment. Most significantly, the vital proton pumping subunits of the respiratory complexes (I, III, IV) are all coded by the mtDNA. In addition, mtDNA encodes the two proton-conductive subunits of complex V, the F0F1-ATPase. In contrast, the respiratory chain enzyme complex II is entirely coded by nDNA and does not pump protons across the inner mitochondrial membrane. The circular, bacterial-plasmid-like mtDNA is densely packed and lacks introns and an effective repair system. Located in the matrix, it is exposed to radical oxygen species (ROS) generated during mitochondrial respiration. ROS can cause point mutations in mtDNA leading to deletions and release of mtDNA fragments, some of which are incorporated into nDNA. It has been proposed that such fragments might cause cancer. Extra- mitochondrial DNA can enter mitochondria via porin, a possible entry point for introducing therapeutic DNA. Transfection of old or uncoupled mitochondria reduces cell viability in vitro. NCL = neuronal ceroid lipofuscinosis. factor (mtTFA), cytochrome c1, adenine nucleotide was increased. Nuclear OXPHOS gene transcripts were β also increased including the ATP synthase β subunit, translocator 2, and F1-ATPase subunit were trans- fected into the Drosophila cell lines. All 4 promoters the heart-muscle isoform of the adenine nucleotide harbor multiple, proximal Sp1-activating elements that translocator, and subunits for Complex I. Besides, account for half or more of transcription activation ancillary nuclear gene transcripts were increased by Sp1, regulating both positive and negative expression including muscle mitochondrial creatine phospho- of the nuclear genes that code for OXPHOS subunits. kinase, hexokinase I, the E1α subunit of pyruvate A study of human tissues containing 12% or less dehydrogenase, muscle glycogen phosphorylase, and non-mutant mtDNA revealed increased transcript phosphofructokinase [172]. There is evidence that levels of a variety of oxidative phosphorylation and transcription of nuclear-encoded molecules that related bioenergetic genes. The patients suffered either support mtDNA replication is unaltered even by from myopathies, encephalopathy, lactic acidosis, substantial variations in mtDNA levels [176]. stroke-like episodes (MELAS), or other pathogenic 3. Mitochondrial DNA. (a) Inhibition of mtDNA mtDNA mutations. The level of mtDNA transcripts replication. Azidothymidine (3'-azido-3'-deoxythy- Molecular pathology of the mitochondrion 41 midine, AZT, zidovudine) and other antiviral deoxyguanosine (dG) to 8-OH-dG, indicating ROS nucleoside analogue drugs inhibit mitochondrial DNA damage. It was suggested that lack of a mitochondrial replication [177]. They are toxic to muscle mitoc- DNA-repairing system enhanced the damage to hondria. AZT is a DNA chain terminator, which mtDNA caused by AZT. The hypothesis was that the inhibits the mitochondrial γ-DNA polymerase that is combined effect resulted in impaired mitochondrial required for mitochondrial DNA replication [178, respiratory chain function causing oxygen radicals that 179]. Long-term treatment with AZT induces delayed were responsible for 8-OH-dG formation [185]. and sometimes severe mitochondrial toxicity [180]. Azidothymidine reduces the activities of Complexes For instance, Southern blotting of muscle biopsy I and III of the mitochondrial respiratory chain. After specimens of AZT-treated patients with myopathy supplementation with AZT of human muscle cells in revealed an up to two-thirds reduction in vitro their mitochondria were enlarged and contained mitochondrial DNA compared with normal adult electron-dense deposits in the matrix and abnormal controls. However, cessation of the treatment reversed cristae. The respiratory control ratio was decreased the depletion of mtDNA [181]. indicating uncoupling [186]. Rats treated with AZT It has been argued that either infection with the showed increased serum lactate and glucose levels and human immunodeficiency virus (HIV) or 100-fold elevation of creatine kinase. The highest tissue azidothymidine treatment can cause myopathy [182, concentration of AZT was found in the skeletal muscle 179]. One study postulated that HIV infection rather and the heart [186]. Other findings indicate that inhib- than the AZT (ZDV) caused the myopathies in the ition of electron transport chain enzyme complexes is majority of the patients [183]. However, several other tissue-specific. In vitro, AZT inhibited Complex I in studies indicate that typical mitochondrial mitochondria isolated from liver, skeletal muscle, and abnormalities are observed only in biopsies of AZT- brain. In addition, in this study it inhibited Complex treated patients but not in mitochondria from the non- II of mitochondria isolated from the muscle [187]. treated HIV positive patients [182, 179]. In one such 4. Abnormal mitochondrial degradation. Lipo- study, the abnormal mitochondria contained para- fuscin, the “wear-and-tear” aging pigment found in crystalline inclusions and were found in “ragged-red” the heart and other organs of older individuals, fibers of the biopsy specimen [182]. probably develops from dead mitochondria. With Azidothymidine has also been employed to develop increasing age, the yellow brown pigment accumulates a rat model of the mitochondrial energy decline around the nucleus of the postmitotic cell. In vitro associated with overt mitochondrial diseases and the experiments using isolated mitochondria suggest that aging process [184]. Treatment of the rats with AZT can develop from mitochondria by lipid induced a decline in soleus muscle function in vivo peroxidation: the conversion could be prevented by and ex vivo and decreased bioenergetic capacity of heart addition of an antioxidant to the incubation medium sub-mitochondrial particles. When such particles were [188]. Furthermore, electron microscopy findings of prepared from heart mitochondria of young and aged old myocardium have identified mitochondrial rats treated with AZT, the mitochondrial particles membrane fragments in lipofuscin granules. The derived from the aged rats were less able to maintain fragments have been tentatively identified as remnants the membrane potential, compared to those prepared of mitochondrial cristae, supporting the notion that from the young rats. Remarkably, treatment with Q10 the lipofuscin is derived from dead mitochondria in improved soleus muscle function in vivo and the postmitotic cell [189]. In vitro exposure to 40% significantly improved cardiac mitochondrial ambient oxygen or inhibition of proteases with the thiol membrane potential capacity in vitro [184]. protease inhibitor leupeptin caused accumulation of Damage to mtDNA by oxygen radicals is the fluorescent material consistent with ceroid and primary cause of mitochondrial myopathy with AZT lipofuscin within the secondary lysosome of cultured therapy. A 4-week period of administration of low AG-1518 human fibroblasts. The findings suggest that doses of AZT to mice caused the mouse liver ceroid and lipofuscin form through peroxidative mitochondria to convert one quarter of the total damage of autophagocytosed material [190,191]. 42 Annals of Clinical & Laboratory Science

Mitochondrial energy conversion. 1. The membrane. (b) Hyperpolarization. Hyperpolarization of the (a) Conductivity. Membrane conductivity depends inner mitochondrial lipid-bilayer membrane leads to upon the type of lipid in the membrane as well as the a large, non-linear increase in proton permeability type and concentration of membrane protein [192] [195], similar to the proton permeability of pure (Figure 5). The composition of fatty acids of the phospholipid bilayers. In vitro findings suggest that membrane phospholipids affects the permeability of the lipid-dependent proton leak accounts for only the membrane [193]. The effect of dietary lipids and about 5% of the total mitochondrial proton leak [196]. mitochondrial function are of particular interest, since In addition, the proton leak is significantly modified it has been found that mitochondrial enzyme activities by the presence of proteins embedded in the are affected both by alterations of mitochondrial mitochondrial membrane [197]. Membrane hyper- membrane lipids as well as mitochondrial enzymes. polarization through ATP hydrolysis by F0F1-ATPase Examples of important membrane dysfunctions are enhances generation of reactive oxygen species. For an increase in the intrinsic rate of proton leakage across instance, isoprenoid farnesol induces ATP hydrolysis the inner mitochondrial membrane, a decrease in in mitochondria in cells of Saccharomyces cerevisiae. It membrane fluidity, or a decrease in cardiolipin, the inhibits oxygen consumption and induces the proton protein that supports the function of many inner pumping function of F0F1-ATPase, which results in membrane enzymes. Serious alterations of these mitochondrial inner membrane hyperpolarization. functions may be caused by the synthesis of defective The F0F1-ATPase inhibitor oligomycin and the F1- mitochondrial lipids or proteins, by a decrease in the ATPase inhibitor sodium azide abolish the effect [198]. synthesis of lipids or mitochondrial proteins or the (c) Cardiolipin. Cardiolipin is synthesized in synthesis of faulty proteins, or by increased turnover mitochondria and is localized almost exclusively within of mitochondrial lipids or proteins [194]. the inner mitochondrial membrane. It is a phospho- lipid (disphospatidylglycerol). Cardiolipin interacts with membrane-bound proteins to orient and activate them. It affects matrix proteins, mitochondrial membrane receptors, and leader peptides. Cardio- lipins, especially those with high linoleic acid (18:2) content, strongly bind many mitochondrial carrier proteins and oxidative phosphorylation enzymes. They exhibit an especially high affinity for cytochrome oxidase. Whereas cardiolipins are not absolutely essential for activation of this enzyme complex in vitro, maximal activities of cytochrome oxidase are only obtained when cardiolipins are present [199,200]. In vitro, saturated cardiolipins form membrane Fig. 5. Illustration showing causes of dysfunction of the bilayers while unsaturated cardiolipins form inner mitochondrial membrane. Its bilipid structure with nonlamellar phases. Cardiolipins are capable of the hydrophobic center and hydrophilic outsides provides participating in proton conductive pathways along capacitance that permits it to be charged with an electric bilipid membranes when embedded in an ordered potential. The interaction between membrane lipids and bilipid matrix containing phosphatidyl choline and proteins has been studied using alternating current at 1 Hz phosphatidylethanolamine, such as the inner and 1KHz to investigate in vitro both the resistance and membrane of the mitochondrion [201]. capacitance of a lipid bilayer membrane made from either Anti-cardiolipin antibodies are independent risk phosphatidylinositol or oxidized cholesterol. Such measurements have shown that membrane conductivity factors for atherosclerotic vascular disease [202]. depends upon the type of lipid in the membrane as well as Infection with the herpes simplex virus (HSV) can the type and concentration of membrane proteins. For markedly inhibit the synthesis of cardiolipins while details see text. leaving the synthesis of other phospholipids relatively Molecular pathology of the mitochondrion 43 unaffected [203]. Extracts of diesel exhaust particles cases occur in children. Interestingly, a majority of decrease mitochondrial cardiolipin, induce uncoupling, these defects are caused by mutations in nuclear DNA. lower the membrane potential and ATP levels, and As an example, a muscle biopsy from a 10-year-old induce apoptosis [204]. female with arthrogryposis multiplex congenita 2. The electron transport chain (Figure 6). (a) The (Guérin-Stern syndrome) and mild myopathy revealed enzyme complexes of the mitochondrial inner membrane. only half the normal specific activity of Complex I. Most of Complexes I and III and up to 4 copies of The defect was in part compensated for by an increased Complex IV are arranged together in supramolecular number of mitochondria [37]. structures referred to as respirasomes [205] or supra- Human cocaine abuse is associated with heart and complexes [206]. Complex I (NADH:CoQ- liver toxicity. In vitro exposure of neonatal rat cardio- oxidoreductase) is a pyridine nucleotide transhydro- myocytes to cocaine induced slight leakage of lactate genase and a proton pump. It couples the transfer of dehydrogenase and significantly inhibited glutamate/ + + hydride between NADP and NAD to proton trans- malate-mediated respiration of isolated mitochondria, location across the mitochondrial membrane [207]. suggesting inhibition of Complex I [210]. Norcocaine, Complex IV, cytochrome c oxidase, is the third respir- norcocaine nitroxide, and N-hydroxynorcocaine, the atory chain proton pump. It catalyzes the reduction N-oxidative metabolites of cocaine, but not cocaine, of oxygen to water. Its active site consists of a heme deplete ATP of isolated mouse liver mitochondria in group with a binuclear center of a copper ion [208]. vitro. Norcocaine could completely inhibit mitochon- Isolated Complex I deficiency is one of the most drial respiration [211]. common defects of the respiratory chain [209]. Most A mutation at base pair 3,243 in MELAS disrupts transcription of a termination sequence located with the tRNA (Leu) [UUR] gene leading to the synthesis of an abnormal 16S ribosomal RNA. It causes defective translation of ND1-7, the mtDNA-encoded subunits of respiratory Complex I, and alters its affinity for the NADH substrate and reduces the mitochondrial membrane potential. Increased mitochondrial NADH might partially compensate for the defect but requires an intact membrane potential for transport of hydrogen from cytosolic NADH into the mitochondrion. Interestingly, a 5-month course of oral nicotinamide administered to a patient with this MELAS mutation resulted in a 50% fall in blood lactate plus pyruvate concentration [212]. Fig. 6. Schematic illustration of the energy flow in the (b) Coenzyme Q10 - the electron carrier. Coenzyme electron transport chain. The proton-pumping complexes Q10 (Q10), also known as ubiquinone-10, is an I, III, and IV generate the inner mitochondrial membrane endogenously synthesized vitamin-like lipid (Figure 7), ∆Ψ potential, m, typically measuring about 150 mV. an antioxidant, and an essential electron carrier in the Coenzyme Q10 (Q10) accepts electrons from complexes I mitochondrial respiratory chain [213]. Orally and II and transfers them to complex III. Cytochrome c administered it is absorbed to a significant degree. As transfers electrons from complex III to complex IV where an example, supplementation in a randomized the electrons are transferred to oxygen. The mammalian crossover study by Q10 at concentration of 30 mg/ respiratory chain enzymes are not randomly distributed in day resulted in significant increases in serum Q10 levels the mitochondrial inner membrane but arranged in supramolecular clusters referred to as supramolecular whether mixed with the food or administered as a structures or respirasomes. Listed in Italics are agents that capsule [214]. inhibit (below membrane) or stimulate (above membrane). In the blood, Q10 is carried in the circulation in AA: arachidonic acid. For details see text. LDL particles. Inhibition of LDL uptake by a receptor 44 Annals of Clinical & Laboratory Science

shared pathway from acetyl-CoA via farnesyl, the administration of a statin-type of cholesterol-lowering drug that inhibits HMG-CoA reductase reduces the synthesis not only of cholesterol, but also of Q10, and lowers the blood level of Q10 [216]. The results from studies on the regulation of expression and activity of HMG-CoA-reductase, a 97- kDa-protein [217], and the rate-limiting enzyme of the cholesterol biosynthetic pathway are conflicting. As examples, one study found that ATP and insulin stimulate rat hepatic microsomes HMG-CoA- reductase activity, and the activity was 4-fold higher at night compared to daytime activity [218]. However, Fig. 7. This illustration shows the chemical structure of others report that ATP inhibits [95] or inactivates [96] Coenzyme (Co) Q10 (ubiquinone) and part of the synthetic the enzyme. ATP at physiological concentration causes pathway. The polyisoprenoid lateral chain of CoQ10 swift and irrevocable inactivation of the enzyme activity originates from acetyl-CoA that is converted by 3-hydroxy- in the cultured digitonin-permeabilized ovary cell of 3-methylglutaryl coenzyme A (HMG-CoA) reductase to the Chinese hamster [217]. mevalonate, which is then converted to farnesyl. Next, (d) Coenzyme Q10 - inhibition by statins. The statin farnesyl is converted either to cholesterol or CoQ10. types of cholesterol-lowering drugs that inhibit HMG- Administration of a statin-type cholesterol-lowering druginhibits HMG-CoA reductase and reduces the synthesis CoA reductase also reduce synthesis of ubiquinone and of cholesterol and ubiquinone. For details see text. lower the blood level of Q10 [216,219] (Figure 8). The serum Q10 level is also decreased by low-density lipoprotein (LDL) apheresis, and in patients with non- or ligand defect therefore might make a cell more insulin dependent diabetes mellitus (NIDDM) and dependent upon intracellular synthesis of Q10, since normal cholesterol levels, but elevated in diabetic Q10 is essential for proper mitochondrial electron patients with hypercholesterolemia [220]. When a chain transport. group of NIDDM patients with hypercholesterolemia The daily uptake of Q10 differs depending upon were treated with daily doses of 20 mg of simvastatin, the type of food in the diet. For instance, the daily their Q10 blood levels declined significantly. In uptake from the average Danish diet has been estimated contrast, Q10 blood levels in a similar group of 30 at only 3.5 mg of Q10 per day, and was mainly derived patients supplemented with 100 mg Q10 per day from meat and poultry. A recent study of a group of increased significantly [216] (Figure 8). Greenland Eskimos showed that their serum Q10 levels Statin therapy has been reported to induce toxic were significantly higher than the Danish population. myopathy that might be related to dysfunction of These Eskimos live in a most remote area of Greenland mitochondrial oxidative phosphorylation. Indirect and have a low prevalence of ischemic heart disease. It evidence in support of this hypothesis was obtained in is possible that their high serum levels of Q10 are due clinical studies in a group of 60 hypercholesterolemic to the high Q10 levels in their diet, which is derived patients, of which 40 were treated with statins and the primarily from sea mammals and fish [215]. rest served as controls. Compared with the control (c) Coenzyme Q10 - structure and synthesis. group, the statin-therapy group showed a significant Coenzyme Q10 regulates oxidative phosphorylation increase of the blood lactate/pyruvate ratio. The Q10 and prevents lipid peroxidation [216]. Its serum levels in the statin group fell to 0.75 compared polyisoprenoid lateral chain originates from acetyl-CoA with 0.95 mg/L in the untreated patients [219]. via mevalonate and isopentenylpyrophosphate sharing Experiments with brief ischemia and reperfusion its biosynthetic pathway with cholesterol [216]. in dog myocardium suggest that lipophilic but not Mevalonate is converted to farnesyl, which is converted hydrophilic HMG-CoA-reductase inhibitors enter to either cholesterol or Q10 [216]. Because of the Molecular pathology of the mitochondrion 45

However, in a series of similar experiments, but using only 30 minutes of ischemia, pretreatment with the lipid-soluble simvastatin, but not with pravastatin, significantly reduced the myocardial level of Q10. The ex vivo ADP/O ratio with succinate was significantly reduced in mitochondria only from the simvastatin- treated, and not from the pravastatin-treated rats. Thus the decrease of myocardial Q10 levels induced by cholesterol-lowering therapy with a lipid-soluble, but not a water-soluble, statin might cause worsening of heart mitochondrial respiration during ischemia [224]. A significant, progressive deficiency in blood Q10 levels was reported in patients infected with HIV, with ARC, and with AIDS. Treatment with Q10 led to appreciable and sometimes marked clinical improvement [225]. After interventional cardiac procedures, old patients show inferior recovery Fig. 8. Serum (top and bottom) and blood (middle) Q10 levels of patients with hypercholesterolemia (top and middle) compared to young patients. In the rat model of stress and NIDDM (bottom). Statin therapy lowers Q10 levels. induced by rapid electrical pacing of isolated working However, oral Q10 supplementation restores blood Q10 hearts, pre-stress work performance of senescent hearts levels. Data from the following references: De Pinieux et al was inferior to that of young hearts. Remarkably, [219] (top), Bargossi et al [216] (middle), and Miyake et al pretreatment with Q10 abolished the difference [220] (bottom). between the young and old rat hearts [226]. mitochondria, inhibit Q10 biosynthesis and thereby In rabbits, simvastatin induced mitochondrial mitochondrial electron transport, and depress ATP swelling, autophagic vacuoles, and muscle fiber necrosis generation [221]. In the rat model, administration of in all 6 rabbits, pravastatin (300 mg) in 2, and the water-soluble pravastatin to rats age 35 and 55 pravastatin (100 mg) in none of the rabbits. However, weeks significantly accelerated the age-related decline surprisingly, in spite of the decrease in muscle Q10 in the activity of Complex I of mitochondria from the content, more so in the pravastatin group, and the diaphragm and the psoas major muscles. In mitochondrial swelling observed particularly in the comparison, pravastatin had no significant effect on simvastatin group, the mitochondrial respiratory chain mitochondria from the rat heart and liver, which did enzymes were normal in all groups in this study [227]. not show any age-related diminution of respiratory (e) Coenzyme Q10 - regulation of Q10 synthesis. A function up to age 55 weeks. Based upon these findings study of cultured human skin fibroblasts failed to detect it was suggested that proper diaphragmatic function any feedback regulation by Q10 of 3-hydroxy-3- should be carefully considered when prescribing methylglutaryl-Coenzyme A (HMG-CoA) reductase. pravastatin for the elderly patient [222]. Whereas supplementation of the cells with Q10 in the In contrast, others found evidence of simvastatin medium increased the cellular content of Q10 about 10-fold, it did not suppress HMG-CoA reductase nor and pravastatin inhibition of oxidative phosphorylation 14 in the rat heart after experimentally induced ischemia. the incorporation rate of C- acetate into Q10 [228]. The ATP production per unit oxygen in rat heart This would appear to rule out any involvement of mitochondria ex vivo was decreased after one hour of Q10 in the regulation of HMG-CoA. However, if prior in vivo ischemia. Pretreatment of the rats by regulation of HMG-CoA occurs via ATP, an alternate statins enhanced the decline in ATP synthesis. The interpretation is possible. OXPHOS might have been lipid-soluble simvastatin caused a greater decline than fully saturated with Q10 due to an ample endogenous the water-soluble pravastatin [223]. The effect on myo- supply of Q10. The ATP supply would then have been cardial Q10 levels was not determined. adequate before supplementation, and there might have 46 Annals of Clinical & Laboratory Science

been no reason for a compensatory increase in the activity in both membrane-bound and soluble F1 in expression of the reductase to enhance further Q10 vitro [236]. Succinate may partially protect against synthesis, even after the cellular uptake of a significant these light-induced effects [236]. amount of exogenous Q10. The F0 proton-conductive part of F0F1-ATPase (e) Other inhibitors. The poliovirus can significantly conducts protons through the mitochondrial inner alter mitochondrial function as demonstrated by membrane into the matrix. The membrane energy inhibition of Complex II of cultured COS-1 and T47D released is used by the F1 moiety to convert ADT to cells [30]. Ceramide mediates the effects of tumor ATP. The phosphorylation step responds rapidly to necrosis factor-alpha (TNF-γ). It might be involved alterations in the cellular energy demand by adjusting γ in TNF- induced apoptosis since exposure to N- the rate of the intramolecular rotation of F0F1 [174]. acetylsphingosine, a C2-ceramide analog, rapidly Mutations of a gene coding for an OXPHOS inhibits Complex III in isolated mitochondria [229]. enzyme subunit may alter the sensitivity of the enzyme In the rat model, methamphetamine or 3,4-methylene- to inhibitors. As an example, the Sprague-Dawley and dioxymethamphetamine inhibits expression of the BHE/Cdb rat differ in their mtDNA sequence for Complex IV as evidenced by a rapid decrease in a subunit of Complex V. Base substitutions in the cytochrome c oxidase staining in the substantia nigra, ATPase 6 gene result in the substitution of aspartate the nucleus accumbens, and the striatum [230]. for asparagine at position 101 and the substitution of A sufficient supply of iron is necessary to prevent leucine for serine at position 129. As a consequence, dysfunction of iron-containing OXPHOS enzymes. isolated mitochondria from the rat with the base 31P magnetic resonance spectra of the gastrocnemius substitution are more sensitive to oligomycin inhibition muscle of iron-deficient Wistar rats revealed slow of OXPHOS compared with mitochondria isolated recovery of intracellular phosphate and phospho- from the Sprague-Dawley rat. These results are creatine concentration after exercise. The alteration consistent with results from human fibroblasts having in the muscle bioenergetics persisted beyond treatment a mutated ATPase-6 gene [237]. with iron and complete correction of the anemia suggesting impaired oxidative phosphorylation [231]. (f) Aging. Neither Sprague-Dawley rats nor C57/ B17 mice lived longer on a diet supplemented daily with 10mg/kg of Q10 compared to control animals not receiving Q10 supplementation. In the rat, plasma and liver Q10 levels were significantly elevated by the supplementation. However, tissue levels of Q10 in the heart, brain, or kidney were unaffected [232].

3. Phosphorylation. (a) Complex V - the F0F1- ATPase. The final step of the mitochondrial energy conversion is the phosphorylation of ADP (Figure 9).

Complex V, the F0F1-ATPase, provides approximately 95% of the adenosine triphosphate (ATP) produced by the cell [18]. It has been estimated that 3 protons Fig. 9. A schematic illustration of phosphorylation, uncoupling protein (UCP), and the mitochondrial are used by F0F1-ATPase for each ATP molecule that is synthesized [233]. Complex V is inhibited by permeability transition pore complex (PTPC). Complex V, the F F -ATPase, utilizes the membrane potential to phenothiazines and similar compounds [234], and by 0 1 phosphorylate adenosine diphosphate (ADP) to adenosine di-(2-ethylhexyl)phthalate (DEHP), a plasticizer [235]. triphosphate (ATP) and provides most of the ATP produced The anesthetic n-butanol and tetracaine can inhibit by the cell. The adenine nucleotide transporter (ANT) ATPase activity. Irradiation with ultraviolet light results exchanges the ADP and ATP across the membrane. Agents in a conformational change of amino acid residues in that interfere with normal functions are indicated in italics. the active site of ATPase and inhibition of ATPase For details see text. Molecular pathology of the mitochondrion 47

(b) The adenine nucleotide transporter (ANT). The mRNA for cytochrome c1 is increased as much as 20- transmembrane transport of adenine nucleotides by 50-fold by thyroid hormone. However, other nuclear β ANT can be inhibited by morphine [238]. Specific OXPHOS genes such as the F1-ATPase -subunit and antibodies against ANT can limit oxidative the core protein 1 of Complex III do not respond to phosphorylation by inhibiting transmembrane thyroid hormone [245]. Mitochondria from thyroxin- nucleotide transport. Recent findings suggest that a treated rats incubated with succinate as substrate virus might induce cardiac mitochondrial dysfunction showed an ex vivo increase in the respiration rate of via an antibody-mediated attenuation of the capacity almost 50%, and there was a 10% increase in the of ANT to exchange nucleotides. First, immunizing membrane potential compared with mitochondria guinea pigs with ANT disturbs heart mitochondria and from normal, non-treated rats [246]. reduces heart function. Second, infecting mice with In hyperthyroidism, triiodothyronine induces the Coxsackie B3 virus induces specific anti-ANT increased ATP consumption in the heart. However, antibody production in over 70% of the mice. relatively more membrane energy is used for heat Perfused, isolated hearts from the animals that production and less directed toward ATP production produced the anti-ANT antibody showed significantly and muscle contraction [247]. reduced mitochondrial oxidative phosphorylation and Anabolic-androgenic steroids can affect respiratory over 50% reduction in left ventricular pressure [239]. chain enzymes. For instance, there was a significant Bile acids reduce ANT in vitro [240]. decrease in the activities of Complexes I, III, and IV The carboxyl end of the HIV-1 viral protein R binds in rats treated with either fluoxymesterone, methyl- to purified ANT and rapidly dissipates the membrane androstanolone, or stanozolol [248]. Others have potential of isolated mitochondria and induces reported that glucocorticoids such as hydrocortisone, apoptosis in cultured cells. Addition of Bcl-2, which prednisolone, dexamethasone, and triamcinolone, inhibits the opening of the permeability transition inhibit Complex IV [249]. complex pore, prevents the apoptosis [241]. In the animal model the effect of cannabinoids is (c) Regulation of oxidative phosphorylation. Oxidative different for single use compared with prolonged use. phosphorylation is controlled differently in different A single dose (10 mg/kg) of δ(9)-tetrahydrocannabinol tissues. In muscle and heart, control of respiration is increased both oxidative phosphorylation of rat brain the primary control, whereas in brain, liver, and kidney, mitochondria and cerebral lipoperoxidation ex vivo. control is through regulation of the ATP synthase and When the same dose was administered twice daily for the phosphate carrier [242]. 4.5 days, it resulted in uncoupling of brain oxidative One theory of the regulation of oxidative phos- phosphorylation and induced neuronal damage [250]. phorylation is that breakdown products of ATP diffuse 4. Uncoupling. (a) Effects on cell survival. Mitochon- freely to the mitochondria to stimulate OXPHOS. drial transfer experiments have demonstrated that However, calcium entry into the mito-chondria cannot damaged mitochondria can reduce the viability of a explain the first fast phase of oxidative phosphorylation cell [251]. For instance, 20% of young human activation, and it has been proposed that delay of the fibroblasts injected with isolated mitochondria from energy-related signal in the cytoplasm dominates the old rats exhibited signs of degeneration after a few days response time of OXPHOS [243]. [252]. In contrast, only 5% of cells microinjected with Thyroid hormone enhances oxidative phosphory- fresh mitochondrial preparations from young rats lation as shown by thyroidectomy in the rat model of showed signs of degeneration. Young mitochondria hypothyroidism. Removal of the thyroid reduces have a high respiratory control ratio: The energy ATPase activity by over 30% in the liver mitochondria released during mitochondrial respiration is tightly [244]. In the rat, thyroid hormone up-regulates the coupled to phosphorylation of ADP. However, if young expression of selected nuclear genes that encode mitochondria are uncoupled by exposure to an OXPHOS complex sub-units [245]. Even subunits uncoupler such as 2,4-dinitrophenol, the synthesis of of the same enzyme complex may be differentially ATP is reduced. Remarkably, young mitochondria that regulated by thyroid hormone. For instance, the are partially uncoupled induce changes in the recipient 48 Annals of Clinical & Laboratory Science cells similar to those induced by old mitochondria through respiration. Instead of being used for ATP [252]. In such mitochondrial transfer experiments, synthesis the mitochondrial membrane energy is the proportion of dead cells is dependent on the state converted to heat [260]. The heat production not only of uncoupling of the injected mitochondria. On the protects against cold environments but also regulates other hand, supplying the recipient cells with a the mitochondrial energy balance [261]. Through the substrate that is easily metabolized protects the cells. regulation of uncoupling [262], mitochondria can For instance, supplementation with D(-)-beta- adjust their metabolism to the supply of substrates and hydroxybutyrate sodium salt in a dose-dependent the cellular ATP requirement, while minimizing ROS manner prevents degeneration induced by micro- production by lowering the membrane potential [103]. injection of uncoupled mitochondria. The first uncoupling protein (UCP) that was Fatty acids cause non-shivering thermogenesis in discovered is a classical example of an augmentative larger mammals by inducing uncoupling proton flow component. It is a 32-kDa membrane-protein that is across the inner mitochondrial membrane, only above specifically induced in rat brown adipose tissue [263]. a threshold membrane potential of 125 mV. [253]. It was later renamed UCP1, as additional protein The branched-chain phytanic acid, 3,7,11,15-tetra- homologues, namely UCP2, widely expressed in rodent methylhexadecanoic acid, which accumulates through- and human tissues [263], and UCP3 were identified. out the body in Refsum disease, increases the mobility UCP1 uncouples mitochondria of brown adipose tissue of inner membrane phospholipids, alters the conform- [264]. Purine nucleotides on the cytosolic side of ational state and mobility of transmembrane proteins, UCP1 inhibit its proton conductance. Fatty acids and induces uncoupling in vitro [254]. increase UCP1-induced energy dissipation [265] and (b) Passive proton leak. Transmembrane outward regulate the mitochondrial energy system by tuning pumping of protons from the mitochondrial matrix the degree of coupling of oxidative phosphorylation to the intermembranous space and inward transmem- [266]. Retinoids induce UCP1 transcription in brane proton leak results in a futile proton cycle. It transgenic mice [263]. dissipates the mitochondrial redox energy, which In the mouse, LPS, IL-1β, and TNF induce the consumes approximately 15% of the standard expression of UCP2 mRNA. However, the effect is metabolic rate of working muscle and liver in vivo tissue dependent and apparently differently regulated. [255,256]. The proton leak is a basic component For instance, LPS strongly induces the UCP2 present in all mitochondria. expression in muscle and liver, but prior administration Hyperthermia can increase proton conductance of of indomethacin inhibits expression only in liver [267]. the mitochondrial inner membrane and degrade The UCP3 gene is expressed in rodent skeletal oxidative phosphorylation. The resulting proton leak muscle and brown adipose tissue and might also play is caused by alteration of the membrane order. An in a role in the mitochondrial degradation of fatty acids vitro study of intact female rat mitochondria using [261]. Two UCP3 RNA transcripts produce 2 fluorescent measurements to determine membrane isoforms, one long, full length (UCP3L) and one short, phospholipid polarization revealed that the transition truncated (UCP3S) isoform, that are highly expressed occurred between 40 and 43ºC [257]. in skeletal muscle. Both strongly impair the In addition, an augmentative component present mitochondrial coupling efficiency and increase in some mitochondria induces a significant flow of thermogenesis, with the short isoform being the most protons across the mitochondrial inner membranes active [268]. A mutation of the UCP3 gene that into the mitochondrial matrix. Some suggest that in increases the proportion of the short isoform slows its the rat resting hepatocyte almost one-third of its resting insertion into the mitochondrial inner membrane, oxygen consumption is dissipated as heat due to proton decreases fat oxidation, and enhances the susceptibility leaks [258]. The proton leak in rat tissues varies to obesity [269]. depending upon the tissue examined [259,255]. The absence of UCP-1 in the UCP1-ablated mouse (c) Active proton leak. Uncoupling proteins regulate leads to high expression levels of UCP2 and UCP3 the dissipation of the membrane potential formed and induces low cold tolerance. Interestingly, the mice Molecular pathology of the mitochondrion 49 do not become obese, and in these experiments, UCP2 been suggested that onset of MPT might be involved and UCP3 were not associated with any inherent in chemical toxicity and Jamaican vomiting sickness uncoupling or with an increased basal metabolism which, like Reye’s syndrome, are characterized by [264]. In comparison, a 24-hour starvation period in hyper-ammonemia, hypoglycemia, microvesicular the regular rat increased UCP2 and UCP3 in the steatosis, and encephalopathy [273]. skeletal muscle more that 4-fold and doubled the Low temperatures markedly decrease the resting UPC3 protein levels while mitochondrial proton respiration due to membrane leak; however, the conductance remained constant [270]. respiration due to intrinsic proton pump uncoupling The adenine nucleotide translocator (ANT) and increases [274]. In rat liver mitochondria, the glycoside the voltage-dependent anion channel (VDAC) are part antibiotic sporaviridin uncouples oxidative phosphor- of the permeability transition pore complex [241]. ylation. It increases the permeability of the inner During a mitochondrial permeability transition membrane of the mitochondria [275]. Regulating the (MPT), the complex opens the high conductance pore proton leak may rapidly permit mitochondria to that increases membrane permeability to solutes of channel energy flux to or from ATP synthesis [193]. molecular mass up to 1.5 kDa [27]. The mitochondria 5. Functional equivalency diagram. The function swell, the membrane depolarizes and its potential of the oxidative phosphorylation system can be dissipates; oxidative phosphorylation is uncoupled. simplified by reference to a schematic diagram where The onset of a mitochondrial permeability the function of the electron transport chain is compared transition can be observed with confocal fluorescence to that of a fuel cell (Figure 10). The input to the fuel microscopy by using red fluorescing tetramethyl- cell consists of the food metabolites and oxygen; the rhodamine methylester as a membrane potential- energy released charges a capacitor (the membrane). indicating fluorophore in combination with The energy stored temporarily in the capacitor is either cytoplasmic calcein [271]. As pores open, green- coupled to an ATP converter or uncoupled due to fluorescing calcein moves into mitochondria. There inhibition of the converter or due to heat loss caused is a simultaneous release of the red dye. Remarkably, by fixed or variable discharge of the capacitor. The cyclosporin A blocks the opening of the pores and mitochondrial permeability transition (MPT) is prevents cell death. In vitro, salicylate induces the analogous to a switch that rapidly discharges the mitochondrial permeability transition. It kills cultured capacitor. If the switch is closed only momentarily rat hepatocytes at concentrations of 0.3-5 mM in a the fuel cell might rapidly respond and recharge the concentration-dependent manner [27,272]. capacitor. However, if the fuel cell is damaged, Similarly, other compounds, such as 3-mercapto- complete discharge might occur. propionic and 4-pentenoic acids, adipic, benzoic, isovaleric, and valproic acids, and Neem oil that have Mitochondrial Genetics been implicated in the pathogenesis of Reye’s syndrome can induce the onset of the mitochondrial permeability The genome. 1. Phylogenesis. Mitochondrial DNA transition in freshly isolated hepatic mitochondria in sequence data suggest that animal and fungal vitro. Surprisingly, the induction of the MPT in these mitochondrial DNA share a common ancestor. experiments did not substantially reduce the membrane However, the fungal mitochondrial genomes contain potential. This might be due to a rapid increase in a large number of introns, which are absent in human respiration that temporarily maintains the membrane mtDNA [276]. As a result, a deletion of part of the potential [273]. Apparently, an intact, rapidly human mitochondrial genome results in the loss of a responding OXPHOS regulatory system might coding region resulting in defective mitochondrial maintain the inner membrane potential temporarily, protein synthesis of one or more of 13 oxidative phos- even in the presence of some degree of MPT. Such a phorylation enzyme subunits. According to the mechanism might be very important to cell survival, classical serial endosymbiosis hypothesis, mitochondria since these findings suggest that uncoupling alone does are derived from the capture of an α-proteobacterial not always induce complete depolarization. It has also endosymbiont by a nucleus-containing eukaryotic host 50 Annals of Clinical & Laboratory Science

genes code for the proton-pumping subunits of electron transport chain enzyme complexes. As hydrophobic proteins they could not be synthesized in the cytoplasm and imported into mitochondria. Many additional bacterial genes present in the nuclear genome may have originated from bacteria that were simply taken up and processed as food [282]. Some sequence data suggest that the formation of the mitochondrion occurred simultaneously with the formation of the nuclear genome of the eukaryotic cell [277]. Phylogenetic DNA sequence comparisons suggest that extensive lateral gene transfer occurred Fig. 10. Simplified schematic diagram of oxidative between early bacteria, archaea, and ancestral phosphorylation. The electron transport chain is compared eukaryotes, resulting in “chimeric” genomes containing to a fuel cell with an input side consisting of food metabolites genes from multiple sources [283]. and oxygen (not shown) and an output side that charges a 2. The structure. The Cambridge sequence was capacitor (the membrane) up to about 150 mV. The voltage mainly derived from a single human placenta with a is coupled either to (a) an ATP converter or (b) dissipated as heat. Uncoupling is the reduction of ATP generation few regions coming from HeLa cell mtDNA and due to either (a) inhibition of the converter or (b) fixed or bovine mtDNA, which were used to establish the base variable heat loss. Activation of the mitochondrial for several ambiguous nucleotides. The Cambridge permeability transition (MPT) is compared to a switch that sequence consists of 16,569 base pairs encoding rapidly discharges the capacitor. When closure of the MPT hydrophobic protein subunits of the mitochondrial switch starts to discharge the capacitor, a fully operational electron-transport system and ATPase: seven subunits fuel cell might rapidly respond to prevent complete of complex I, apocytochochrome b of Complex III, discharge. However, if the fuel cell response is inadequate, three subunits of Complex IV, and two subunits of complete discharge follows (apoptosis). ATPase (Complex V). The remaining mtDNA genes specify 2 mitochondrial ribosomal RNA (RNA), 22 [277]. The ancient anaerobic host, the archae- organelle-specific transfer RNA (tRNA) and genes bacterium, engulfed the respiring symbiont [278], a regulating transcription and replication (D-loop) [11]. proteobacterium, which evolved into the mitochon- All other mitochondrial proteins are coded for by the drion. An analysis of genomic signatures indicates that nuclear genome. the progenitors of animal mitochondria (and various There are 103 to 105 circular mitochondrial DNA primitive eukaryotes lacking mitochondria) were fusion molecules in the human cell [284]. Heteroplasmy is microbes composed of a Clostridium-like eubacterium the simultaneous presence of both normal and mutated and a Sulfolobus-like archaebacterium [279]. mtDNA [20]. The energy-converting metabolism, originally 3. The high mutation rate of mtDNA. The stability bound to the plasma membrane, was relocated to the of the mitochondrial genome and its efficiently intracellular space. This endosymbiosis led to the regulated expression is essential for maintaining the transfer of most genes from the symbiont to the nuclear membrane potential and a functional oxidative genome of the host [280]. Evolution might have phosphorylation pathway [285]. However, the rate of favored the transfer of bacterial DNA to the more mutation of mtDNA, caused, for instance, by radical protected environment of the nuclear genome to oxygen metabolites produced by the respiratory chain, prevent defective mitochondria from flourishing to the is 10 to 20 times higher than the mutation rate of detriment of an organism, particularly in its syncytial nuclear DNA. Besides, the mtDNA lacks effective tissue [281]. repair and contains no histones. A few genes essential to OXPHOS remained in the Mutations of the mitochondrial genome are the mitochondrion during phylogenesis. Some of these most important causes of known inherited and Molecular pathology of the mitochondrion 51 acquired genetic OXPHOS deficiency [145]. A uncoupling and immature spermatozoa. The degree comparison of the evolutionary rate of the nDNA- of uncoupling also correlated with spermatozoa encoded β subunit with that of the mtDNA encoded defective for nuclear maturity. A higher percentage of ATPase 6- and 8-subunits, 7 other mtDNA OXPHOS immature spermatozoa was found in men from barren genes, and a number of nuclear genes revealed couples compared to donors of proven fertility. significant differences in their synonymous substitution In a patient with inherited mitochondrial disease rate. The rate for the ATPase6 and 8 genes was 12 caused by reduced activity of Complexes I and IV the times greater than the rate of the nuclear gene for the sperm motility was reduced compared to normal β subunit. Even more remarkable was the finding that control [290]. Remarkably, in vitro supplementation the mutation rate of the average mtDNA gene was of the medium with pyruvate that enters the respiratory 17-fold that of the nuclear β subunit gene. These high chain at Complex I and succinate that enters at substitution mutation rates and strong selective Complex II tripled the sperm motility compared with constraints of mammalian mtDNA proteins explain only 12% motility when supplemented with glucose why mtDNA mutations result in a dispropor-tionately alone. Ultrastructural changes of the mitochondria large number of human hereditary diseases of were found not only in the spermatozoa but in sperm- OXPHOS [286]. atids as well, suggesting that the abnormalities were due to a primary mitochondrial defect rather than to Propagation. 1. Mitochondria of the male gamete. The secondary degeneration of the spermatozoa. fate of mitochondrial DNA differs significantly in male Paternal mitochondrial DNA has not been detected and female gametes. Analysis of the entire mitochon- in human somatic cells of the offspring, at least not at drial genome in sperm donors revealed that most of the limits of detection of present technology. It is not the spermatozoan mitochondria from patients with known how the paternal mitochondria are eliminated oligoasthenospermia had multiple mtDNA deletions. from the human morula; however, the process in Surprisingly, multiple deletions were found in normal humans might be analogous to the mechanism in the sperm as well [287]. mouse, which rejects the sperm-derived mitochondria The Darwinian competition to fertilize the ovum at the 4- to 8-cell stage [291]. should favor the sperm with the most efficient 2. Mitochondria and the female gamete. Delayed OXPHOS and therefore the highest motility. There is motherhood is characterized by a higher risk of evidence that factors reducing the mitochondrial energy conceiving an offspring suffering from a mitochondrial production are responsible for some cases of male DNA disorders [292]. And while the mitochondria infertility. For instance, there is a direct correlation from oocytes collected from twelve women showed few between sperm mitochondria respiratory chain enzyme mtDNA rearrangements [287], there was considerable activities and sperm motility. Some findings argue that mutational heterogeneity in the individual oocyte particular cases of asthenozoospermia are caused by donor. However, oocytes have an efficient mtDNA defective mitochondrial energy production. For repair system, which is basically independent of instance, the activities of Complexes I, II, and IV in maternal age [292]. It protects and perpetuates the sperm samples from asthenozoospermic subjects were maternal mitochondrial genome. significantly lower compared with those from the In addition, the process of oogenesis, follicle control individuals [288]. The sperm motility formation, and loss of the less fit purifies the female depended primarily on the mitochondrial volume and germ-line mitochondrial DNA [293]. This culling therefore the total energy available generated by revives the mitochondrial genome as it passes, by way OXPHOS in the sperm midpiece. of the oocyte cytoplasm, from one generation to the findings using the cationic dye next. This process provides the bottleneck that refines JC-1 to measure the sperm mitochondrial membrane the haploid mitochondrial genome in the oocyte and potential corroborate a correlation between the maintains the integrity of maternal mitochondrial potential and sperm motility [289]. Moreover, cyto- inheritance. metry revealed a correlation between mitochondrial 52 Annals of Clinical & Laboratory Science

Cloning. The loss of non-oocyte mitochondria has also introduced to explain this phenomenon. For instance, been detected in mammalian cloning that fused a it has been proposed that sporadic stem cell mutation somatic cell by electroporation to an enucleated oocyte during embryogenesis or mitotic segregation might from the same animal. It might be expected that the result in different degrees of heteroplasmy in various cloned progeny should contain mtDNA from both the tissues [20]. Heteroplasmy can also result from donor and recipient, resulting in heteroplasmy. acquired mutations of mtDNA. However, somatic cells in cloned sheep contain only 3. Mutations and deletions. A defect in the germline the female germ-line mitochondrion [292]. How the mtDNA affects all mtDNA plasmids in all mito- donor cell mitochondria disappear just like sperm chondria of all somatic cells and explains why inherited mitochondria is unknown. Possibly, contrary to the mtDNA diseases can be very serious. Mitochondrial somatic cell mitochondria, oocyte mitochondria might DNA mutations causing overt mitochondrial diseases carry some kind of recognition signal sequence that are characterized by a decline in mitochondrial protects them from expulsion or rapid degradation. respiratory function [184]. This has been demons- As the first cloned sheep showed advanced trated in cases of progressive kidney disease or biological age, it was suggested that this might be due maternally inherited diabetes and deafness, both caused to free radical-induced cellular damage to their by a single point mutation of the mitochondrial tRNA. inherited somatic mitochondria [294]. However, the Cybrid cells were prepared by inserting donor premature aging of the cloned sheep is probably related mitochondria into ρ0 cells, which lack mtDNA. The to their shortened telomeres. Remarkably, the opposite mitochondria were derived from fibroblasts of a patient was observed in cloned cows, where the telomeres in carrying an A to G transition at nucleotide position several cases were elongated, suggesting that cloning 3,243 of the mtDNA (Figure 11). The heteroplasmy can also result in a prolonged life span. in the cybrid cells varied from none to 100%. The cybrid cells containing predominantly mutant mtDNA Genetic diseases. 1. The problem with limited diversity. showed marked defects in mitochondrial morphology, An important point in inherited mitochondrial diseases poor respiratory chain Complex I and IV activities, is the fact that populations with limited genetic and lactic acidosis [296]. diversity are at risk for diseases that are rare elsewhere. Similarly, cybrid clones were used to demonstrate A case in point is the so-called “Finnish disease heritage” mitochondrial respiratory chain dysfunction due to a [295]. An analysis of mitochondrial mutations that single point mtDNA mutation in a case of familial have accumulated revealed that only a small number hypertrophic ventricular cardiomyopathy [297]. A of men and women contributed to the genetic lineage fibroblast cell line derived from the patient carried a present in the Finnish population. The mitochondrial T9997C (Figure 11) mutation of the mtDNA gene genes examined were those of the mtDNA nucleotide encoding tRNA glycine. The cybrid clones were positions that evolve slowly in the mitochondrial obtained by fusion of ρ0 osteosarcoma cells to control region. For the nuclear genes, the Y-chromo- enucleated patient skin fibroblasts. The clones having somal haplotype population of the peroxisome high levels of heteroplasmy of mutant mtDNA showed γ proliferator-activated receptor gamma (PPAR )- mainly Complex I and cytochrome c oxidase deficiency. coactivator-1 (PGC-1) was analyzed [295]. PGC-1 An elevated lactate/pyruvate (L/P) ratio corroborated coordinates the expression of both mitochondrial and the presence of respiratory chain deficiency. nuclear encoded OXPHOS enzyme subunits and the More than 100 primary defects in the mitochon- uncoupling protein [103]. drial genome have been associated with encephalo- 2. Heteroplasmy. In diseases caused by inherited myopathies and the majority has been linked to mutations of mtDNA, the link between genotype and defective oxidative phosphorylation [298]. For phenotype varies so that the same mtDNA mutation instance, several mtDNA mutations cause rare may give rise to a variety of phenotypes. Moreover, the encephalomyopathies such as Leigh and Leigh-like same phenotype may be seen with different mtDNA syndromes, fatal infantile lactic acidosis, neonatal mutations [145]. Differing points of view have been cardiomyopathy with lactic acidosis, and macrocephaly Molecular pathology of the mitochondrion 53

Fig. 11. Mitochondrial deletions (top) of the mitochondrial genome (bottom), which are illustrated in nucleotides (nt) in linear form along the x-axis. For clarity, the mitochondrial genes coding for subunits of oxidative phosphorylation (OXPHOS) enzyme Complexes I, III, IV, and V are shown in separate lanes. Genes that are partially or completely deleted by the ‘common’ deletion are highlighted. The map was prepared from numerical data downloaded from “MITOMAP: A Human Mitochondrial Genome Database. Center for Molecular Medicine, Emory University, Atlanta, GA, USA, http:// www.gen.emory. edu / mitomap.html, 1999”. with progressive leukodystrophy [209]. Knowledge administration of redox agents, vitamins, coenzymes, of the normal mtDNA variation in a specific human and enzyme activator [212]. population is essential to understand the pathophysiol- Antioxidants limit lipid peroxidation and decrease ogy of diseases caused by an mtDNA aberration [299]. prostaglandin synthesis [300]. The risk of oxygen- By comparison, mutated nDNA genes encode derived free radical damage can be significantly reduced mitochondrial OXPHOS proteins that cause by improved diet, particularly fruits, vegetables, and Friedreich’s ataxia and hereditary spastic paraplegia grains, by reasonable exercise, and by reduced alcohol [145]. In the case of amyotrophic lateral sclerosis and consumption [301]. Huntington’s disease, the defect of oxidative phosphorylation is secondary to events induced by a Q10. Therapeutic use of Q10 can benefit patients mutation in a nuclear gene encoding a non- with cardiomyopathy. Determination of Q10 tissue mitochondrial protein [145]. levels in myocardial biopsies from a series of 45 patients with cardiomyopathy using high performance liquid Therapy (HPLC) showed significantly lower levels of the coenzyme in the cases of more advanced Several approaches have been used to treat mitochon- than in the milder cases of heart failure. Oral drial diseases, ranging from dietary measures to supplementation with 100 mg of Q10 daily benefited 54 Annals of Clinical & Laboratory Science nearly two-thirds of the patients. Those with dilated Melatonin enhances the activity of Complexes I cardiomyopathy showed the most clinical improve- and IV and prevents ruthenium-red-induced reduction ment [29]. in the activities of these complexes in vivo, suggesting Administration of Q10 significantly reverses a therapeutic use of melatonin in drug-induced exercise-induced abnormalities of oxidative mitochondrial damage [309]. Melatonin can also phosphorylation of the brain of patients with known attenuate ethanol-induced mitochondrial DNA mitochondrial enzyme defects. Magnetic resonance depletion, which occurs after an alcohol binge [310]. spectroscopy studies of such patients show that exercise However, the lack of purity of commercial increases the level of ADP and inorganic phosphate melatonin preparations limits their use. For instance, and reduces the level of phosphocreatine in the brain. 6 impurities detected in 3 commercial melatonin Treatment with Q10 significantly shortens the rate of preparations are structural analogs of contaminants brain phosphocreatine recovery after the exercise and found in the dietary supplement L-tryptophan. These corroborates in vitro observations that Q10 concent- contaminants were implicated as etiologic agents in ration in the inner mitochondrial membrane regulates the eosinophilia-myalgia syndrome epidemic that the efficiency of oxidative phosphorylation [302]. occurred a few years ago [311]. The liver mitochondria of the diet-induced atherosclerotic rabbit showed a significant increase in Carnitine. Carnitine is essential for long-chain fatty hydroperoxides and a serious drop in the content of acid oxidation and for shuttling accumulated acyl Q10. Virgin olive oil or vitamin E-stabilized fish oil, groups out of the mitochondria [312]. In the chronic but not sunflower oil, added to the diet in part reversed alcohol-fed rat, oral L-carnitine by itself, but more so the diet-induced alterations [303]. Coenzyme Q10 if administered with oral Q10, protects against alcohol- (3 mg/kg/day) administered to rabbits fed an induced hepatic lipid infiltration [313]. atherogenic diet significantly reduced aortic cholesterol In humans, therapy with carnitine ameliorates the and aortic as well as coronary artery plaque size [304]. symptoms of claudication of peripheral arterial disease. When applied to the epidermis, Q10 penetrates into It restores skeletal muscle function of patients on the viable layers. Weak photon emission measurements dialysis who suffer from dialysis-induced reduction of indicate that it reduces the level of epidermal oxidation. muscle carnitine. However, while carnitine supple- It prevents photoaging, suppresses the expression of mentation improves exercise performance in certain collagenase, and reduces the wrinkle depth [305]. disease states, any benefit in healthy individuals to An adequate supply of vitamin B6 is essential for support the high metabolic demands of heavy exercise the endogenous synthesis of the quinone nucleus of is uncertain [312]. coenzyme Q10 (Q10) from tyrosine. It should therefore be recommended that patients treated with Gingko biloba. By scavenging the superoxide anion, Q10 should receive concurrent supplementation with the Ginkgo biloba Egb761 extract provides post- B6 to enhance endogenous synthesis of Q10 [306]. ischemic protection against re-oxygenation injury to rat liver mitochondria in vitro [314]. Besides, Melatonin. A pineal hormone melatonin, N-acetyl- treatment with Egb761 extract in part prevents the 5-methoxytryptamine [307], lowers plasma cholesterol development of peroxide formation in the levels in genetically hypercholesterolemic rats. It also mitochondrion of the old rat [315]. Extract of Ginkgo reduces the fatty changes in their liver [308]. Most biloba is anti-ischemic mainly due to the presence of importantly, melatonin concentrates in mitochondria. bilobalide in the terpenoid fraction. Bilobalide delays In rats fed a 1% cholesterol + 0.5% cholic acid diet hypoxia-induced decrease in ATP content of daily, treatment with ip injections (4 mg) of the endothelial cells in vitro [316]. It increases the activity antioxidant melatonin for periods of up to 4 weeks of Complex I, protects Complexes I and III activities lowered the diet-induced increases in plasma levels of and delays the onset of ischemia-induced damage. It VLDL and LDL. It decreased plasma HDL and permits respiratory activity and ATP regeneration by diminished diet-induced fatty change of the liver [307]. mitochondria under ischemic conditions as long as Molecular pathology of the mitochondrion 55 some oxygen is present [317]. These insights were mutated genomes and restoring health by permitting obtained using mitochondria isolated from rats treated the propagation of only the wild-type mitochondrial with bilobalide (2-8 mg/kg). There was a dose- DNA [322]. dependent increase in the respiratory control ratio, by reason of lower oxygen consumption during state 4. Conclusions Bilobalide reduced the sensitivity of oxygen consumption to inhibition of Complex I by amytal or Mitochondrial medicine began with the description Complex III by myxothiazol or antimycin A. In of Luft’s syndrome in 1962. The field developed rapidly Alzheimer’s disease, therapy with recombinant human after the complete sequence of mitochondrial DNA apoε3/ε3, apoe3/e4, dehydroepiandrosterone was determined in 1981 and the methods of molecular (DHEA), or Ginkgo biloba extract (EGb 761) protected biology were applied to study the role of the mito- against the lipid peroxidation [148]. chondrion in the etiology of human diseases. This unique organelle developed from endocytosed Dietary fats. Consumption of fish oil lowers plasma bacteria. Most of the bacterial genes ended up in the triacylglycerol levels. There is evidence that the effect host cell nucleus that controls the biosynthesis of the is due to increased mitochondrial fatty acid oxidation. present day mitochondrion. Only 37 genes remained As an example, in rat liver parenchymal cells and in as maternally-inherited mitochondrial DNA. A few purified mitochondria, eicosapentaenoic acid (EPA) of these genes code for the lipophilic, proton-pumping increases mitochondrial fatty acid oxidation. It also subunits of Complexes I, III, and IV of the electron increases carnitine palmitoyltransferase-I [318]. transport chain, which establish the mitochondrial Supplementation of the diet with virgin olive oil to transmembrane potential that powers the synthesis of enrich the mitochondrial membranes with mono- ATP. Hence, the expression of both genomes is unsaturated fatty acids and Q10 prevents free radical essential for the proper biosynthesis and function of damage to heart mitochondria of male rats [319]. the mitochondrial oxidative phosphorylation system. Populations with limited genetic diversity have high Genetic engineering. Genetic material can enter risk for rare mitochondrial diseases. Mitochondrial mitochondria in vivo under both physiological and DNA has a much higher mutation rate than nuclear pathological conditions. In vitro, double-stranded DNA, because it lacks histones and is exposed to radical DNA crosses lipid bilayers doped with isolated oxygen species (ROS) generated by the electron mitochondrial porin that serves as a voltage-dependent transport chain, and the mitochondrial DNA repair anion channel. DNA crossing requires application of system is limited. ROS-induced deletion of fragments an electrical field to the membrane and is blocked by of mtDNA promotes premature aging, and migration addition of anti-porin antibody [320]. This discovery of the deleted fragments into the nuclear genome has opens up the possibility of mitochondrial genetic been linked to carcinogenesis. When the amount of engineering by introducing new DNA into the mtDNA with mutation or deletion in a cell reaches a mitochondrion to repair mtDNA defects. Besides, it tissue-dependent threshold, the cellular metabolism might be possible to introduce DNA sequences that becomes critical and the cell undergoes necrosis. might bestow resistance to adult-onset diseases by In specialized tissues, mitochondria perform a preventing obesity and atherosclerosis or premature number of diverse functions, but the common function cognitive decline. For instance, a mitochondrial in all mitochondria is the generation of ATP. The genotype, mt5178A, was recently identified in Japanese electron transport chain carries electrons from centenarians, suggesting that some mtDNA sequences metabolites, which acts as a fuel, to oxygen, and stores might be more resistant to mutation than others [321]. the extracted energy as a membrane potential that is To cure mitochondrial diseases due to mutated used as needed for ATP or heat generation. However, mtDNA, it has been proposed to introduce anti- the oxidative phosphorylation system is vulnerable to mtDNA sequence-specific molecules into the structural and functional damage. For instance, a mitochondrion, selectively inhibiting replication of temporary lack of oxygen caused by ischemia and 56 Annals of Clinical & Laboratory Science reperfusion increases the generation of ROS. The imported, apoE-binding part of Complex V. However, membrane energy stores can be depleted by excessive the second option has not been demonstrated experi- membrane leakage due to damage by ROS or due to mentally. inappropriate membrane lipid content. Inhibition of Oral therapy with α-tocopherol and Q10 helps enzymes by endogenous or exogenous compounds or prevent atherosclerosis by protecting the LDL particle, lack of or inhibition of the electron carrier Q10, ANT, the major carrier of Q10 in the plasma, from or other transporters can reduce ATP generation. peroxidation. Lipoprotein uptake and intracellular Acidic NSAIDs inhibit or uncouple oxidative synthesis provide Q10 for regulation of oxidative phosphorylation and induce the “topical phase” of phosphorylation where Q10 is essential as it transfers gastrointestinal ulcer formation. In vitro, vacuolating electrons from Complexes I and II to Complex III. cytotoxin prepared from H. pylori decreases the Importantly, statins that inhibit HMG-CoA-reductase mitochondrial inner membrane potential of the lower plasma levels of cholesterol and Q10 because cultured gastric epithelial cell. If the bacterium causes Q10 shares part of its isoprenyl side-chain synthesis uncoupling in vivo as well, it might induce the “topical pathway with cholesterol. Oral therapy with Q10 phase,” leading to ulcer formation even when a selective normalizes its plasma levels and as therapy can also COX-2 inhibitor is administered. However, it has not improve cardiac function in cardiomyopathies. yet been shown that H. pylori uncouples mitochondria Much useful knowledge has been revealed by in vivo. Aspirin inhibits β-oxidation, but not electron recent research on oxidative phosphorylation. A few transport. However, the aspirin metabolite salicylate therapeutic alternatives are available. Avoiding decouples mitochondria by inducing the mitochondrial substances that damage mitochondria and supple- permeability transition. Cocaine inhibits Complex I, mentation with compounds that protect mitochondrial the poliovirus inhibits Complex II, ceramide inhibits structure and function are presently most important. Complex III, and azide, cyanide, chloroform, and There is a need to develop genetic engineering methods methamphetamine inhibit Complex IV. By contrast, to repair or replace damaged mitochondrial DNA. melatonin stimulates Complexes I and IV and Gingko biloba stimulates Complexes I and III. 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